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| United States Patent |
6,090,911
|
|
Petka
, et al.
|
July 18, 2000
|
Reversible hydrogels
Abstract
The invention is based on the discovery that a block copolymer that
includes .alpha.-helical blocks, e.g., terminal blocks, which form
intermolecular coiled-coil structures, and one or more random-coil blocks,
which link the .alpha.-helical blocks, can form suspensions that can
reversibly gel to form monodisperse hydrogels. The transition between the
gel and liquid phases depends on pH, temperature, concentration, and
chemical structure. The copolymers can be synthesized biologically through
genetic engineering.
| Inventors:
|
Petka; Wendy A. (St. Paul, MN);
Tirrell; David A. (Sunderland, MA);
McGrath; Kevin P. (Alpharetta, GA)
|
| Assignee:
|
University of Massachusetts (Boston, MA)
|
| Appl. No.:
|
956307 |
| Filed:
|
October 22, 1997 |
| Current U.S. Class: |
530/300; 424/78.02; 424/78.06; 424/445; 530/350 |
| Intern'l Class: |
C07K 014/00; A61K 031/74; A61K 038/00 |
| Field of Search: |
530/300,350
424/445,78.02,78.06
514/2
|
References Cited
U.S. Patent Documents
| 5243038 | Sep., 1993 | Ferrari et al. | 536/23.
|
Other References
Morii, H. et al., Bull. Chem. Soc. Jpn., vol. 64, No. 2, pp. 396-402, 1991.
McGrath et al., "Genetically Directed Syntheses of New Polymeric Materials.
Expression of Artificial Genes Encoding Proteins with Repeating
-(AlaGly).sub.3 ProGluGly-Elements," J. Am. Chem. Soc., 114(2):727-733,
1992.
McGrath and Kaplan, "Self-Assembling Nanostructures: Recognition and
Ordered Assembly in Protein-Based Materials," Mat. Res. Soc. Symp. Proc.,
292:83-91, 1993.
McGrath and Kaplan, "Control of Molecular Organization in Protein-Based
Materials," Polym. Preprints, 34(1):102-103, 1993.
Petka et al., "Surface Recognition and Diffusion of Engineered
Macromolecules," Polymer Preprints, vol. 35(2), pp. 452-453, 1994.
|
Primary Examiner: Achutamurthy; Ponnathapu
Assistant Examiner: Tung; Peter P.
Attorney, Agent or Firm: Fish & Richardson P.C.
Claims
What is claimed is:
1. A synthetic block copolymer XYZ, comprising:
two .alpha.-helical protein blocks X and Z, each having an amino acid
sequence and conformation that allow each .alpha.-helical protein block to
form a coiled-coil with an .alpha.-helical protein block on another block
copolymer XYZ;
a water-soluble, random-coil protein block Y, the random-coil protein block
linking the two .alpha.-helical protein blocks; and
linker proteins that link the .alpha.-helical protein blocks to the
random-coil protein block.
2. A synthetic block copolymer comprising:
at least two .alpha.-helical protein blocks, each having an amino acid
sequence and conformation that allow each .alpha.-helical protein block to
form a coiled-coil with an .alpha.-helical protein block on another
molecule of the block copolymer, wherein each .alpha.-helical protein
block comprises at least 28 amino acids; and
at least one water-soluble, random-coil protein block comprising at least
15 amino acids, the random-coil protein block linking at least two of the
.alpha.-helical protein blocks.
3. A synthetic block copolymer XYZ, comprising:
two .alpha.-helical protein blocks X and Z, each having an amino acid
sequence and conformation that allow each .alpha.-helical protein block to
form a coiled-coil with an .alpha.-helical protein block on another block
copolymer XYZ, wherein each .alpha.-helical protein block comprises at
least 28 amino acids; and
a water-soluble, random-coil protein block Y comprising at least 15 amino
acids, the random-coil protein block linking the two .alpha.-helical
protein blocks.
4. A block copolymer of claim 3, wherein X and Z are identical to each
other.
5. A block copolymer of claim 3, wherein X and Z are non-identical.
6. A block copolymer of claim 3, wherein Y has the sequence [(AlaGly).sub.p
ProGluGly].sub.n (SEQ ID NO: 23), where p is 0 to 4 and n is 5 to 100.
7. A block copolymer of claim 6, wherein n is 8 to 54.
8. A block copolymer of claim 6, wherein p is 3.
9. A block copolymer of claim 3, wherein the sequences of amino acids that
make up X and Z have an (ABCDEFG).sub.m (SEQ ID NO: 24) pattern; wherein m
is 4 to 100; A and D are hydrophobic amino acids; E and G are polar amino
acids; and B, C, and F can be any amino acid.
10. A block copolymer of claim 9, wherein m is 6 to 18.
11. A block copolymer of claim 9, wherein more than 80% of the E and G
amino acids of X are acidic amino acids and more than 80% of the E and G
amino acids of Z are basic amino acids.
12. A block copolymer of claim 9, wherein more than 80% of the E amino
acids of X and G amino acids of Z are acidic amino acids and more than 80%
of the E amino acids of Z and G amino acids of X are basic amino acids.
13. A block copolymer of claim 9, wherein more than 80% of the E amino
acids of X and Z are acidic amino acids and more than 80% of the G amino
acids of X and Z are basic amino acids.
14. A synthetic block copolymer comprising:
at least two .alpha.-helical protein blocks, each having an amino acid
sequence and conformation that allow each .alpha.-helical protein block to
form a coiled-coil with an .alpha.-helical protein block on another
molecule of the block copolymer; and
at least one water-soluble, random-coil protein block, the random-coil
protein block linking at least two of the .alpha.-helical protein blocks,
wherein the water-soluble, random-coil protein block has the sequence
[(AlaGly).sub.p ProGluGly].sub.n (SEQ ID NO: 23), where p is 0 to 4 and n
is 5 to 100.
15. A block copolymer of claim 14, wherein n is 8 to 54.
16. A block copolymer of claim 14, wherein p is 3.
17. A synthetic block copolymer comprising:
at least two .alpha.-helical protein blocks, each having an amino acid
sequence and conformation that allow each .alpha.-helical protein block to
form a coiled-coil with an .alpha.-helical protein block on another
molecule of the block copolymer, wherein the sequence of amino acids that
make up at least one of the .alpha.-helical protein blocks has an
(ABCDEFG).sub.m (SEQ ID NO: 24), pattern; wherein m is 4 to 100; A and D
are hydrophobic amino acids; E and G are polar amino acids; and B, C, and
F can be any amino acids; and
at least one water-soluble, random-coil protein block, the random-coil
protein block linking at least two of the .alpha.-helical protein blocks.
18. A block copolymer of claim 17, wherein m is 6 to 18.
19. A synthetic block copolymer XYZ comprising:
two .alpha.-helical protein blocks X and Z, each having an amino acid
sequence and conformation that allow each .alpha.-helical protein block to
form a coiled-coil with an .alpha.-helical protein block on another block
copolymer XYZ;
a water-soluble, random-coil protein block Y, the random-coil protein block
linking the two .alpha.-helical protein blocks; and
a recognition sequence or other peptidic target sequence that specifically
binds to a macromolecule.
20. A synthetic block copolymer XYZ comprising:
two .alpha.-helical protein blocks X and Z, each having an amino acid
sequence and conformation that allow each .alpha.-helical protein block to
form a coiled-coil with an .alpha.-helical protein block on another block
copolymer XYZ;
a water-soluble, random-coil protein block Y, the random-coil protein block
linking the two .alpha.-helical protein blocks; and
a recognition sequence or other peptidic target sequence that specifically
binds to a cell.
21. A block copolymer of claim 20, wherein the cell is a fibroblast.
22. A block copolymer of claim 20, wherein the recognition sequence
comprises the sequence ArgGlyAsp.
23. A gel, comprising:
a liquid and a block copolymer of claim 3 suspended in the liquid to form a
suspension.
24. A gel of claim 23, wherein the liquid is an aqueous liquid.
25. A gel of claim 24, wherein the aqueous liquid is water.
26. A gel of claim 23, wherein the suspension is monodisperse.
27. A gel comprising:
a liquid and the block copolymer of claim 19 suspended in the liquid.
28. A gel, comprising:
a liquid and a block copolymer of claim 2 suspended in the liquid.
29. A wound dressing comprising a synthetic block copolymer XYZ and an
antibiotic compound, wherein the copolymer and the antibiotic are both
dissolved in a liquid, and the copolymer comprises:
two .alpha.-helical protein blocks X and Z, each having an amino acid
sequence and conformation that allow each .alpha.-helical protein block to
form a coiled-coil with an .alpha.-helical protein block on another block
copolymer XYZ; and
a water-soluble, random-coil protein block Y, the random-coil protein block
linking the two .alpha.-helical protein blocks.
30. A method of dressing an abrasion, burn or non-puncture wound comprising
applying the block copolymer of claim 3 to said wound.
Description
BACKGROUND OF THE INVENTION
The invention relates to a class of block copolymers that form solutions
that can reversibly gel under specific conditions.
Gels are polymer networks that can absorb solvents and thereby swell. For
example, hydrogels are gels that swell in aqueous solutions. In general,
gels fall into two categories. In crosslinked gels, the polymer chains
that make up the networks are covalently bonded to each other. In physical
gels, the polymer chains are attracted to each other by non-covalent
forces (e.g., ionic, electrostatic, and/or van der Waals interactions).
Because the polymer chains of a physical gel are not chemically
crosslinked, physical gels can have special properties. For example,
physical gels can be highly responsive to physical stimuli such as
temperature, pH, ion concentration, solvent polarity, or polymer
concentration. The responsiveness can be manifest as swelling or shrinkage
of the gel, changes in the shape of the gel, or changes in the viscosity
of the gel. The gel can also undergo a reversible phase change between its
gel and liquid states under appropriate conditions.
A class of physical gels that are of particular interest are the
monodisperse gels. Monodisperse gels are made up of identical polymer
chains of uniform size. Because each chain is of the same molecular
weight, the spacing between structural elements is also of generally
uniform size. By appropriate design, more direct control of pore size can
be achieved in a monodisperse polymer system relative to a polydisperse
system.
SUMMARY OF THE INVENTION
The invention is based on the discovery that a synthetic block copolymer
that includes .alpha.-helical blocks, e.g., terminal blocks, which form
intermolecular coiled-coil structures, and one or more random-coil blocks,
which link the .alpha.-helical blocks, can form suspensions that can
reversibly gel to form monodisperse hydrogels. The transition between the
gel and liquid phases depends on pH, temperature, concentration, and
chemical structure. The copolymers can be synthesized chemically and
biologically, e.g., through genetic engineering.
One embodiment of the invention features a synthetic block copolymer XYZ.
The block copolymer XYZ includes two .alpha.-helical protein blocks X and
Z, each having an amino acid sequence and conformation that allow each
.alpha.-helical protein block to form a coiled-coil with an
.alpha.-helical protein block on another block copolymer XYZ; and a
water-soluble, random-coil protein block Y, the random-coil protein block
covalently linking the two .alpha.-helical protein blocks. For example,
the first .alpha.-helical block, the random-coil block, and the second
.alpha.-helical block can form a continuous peptide chain. Optionally,
other amino acid sequences such as .beta.-sheet or turn sequences can be
included in the peptide chain, either at the ends of the chain or between
the other blocks (e.g., between the .alpha.-helical and random-coil
blocks).
Another embodiment of the invention features a synthetic block copolymer
having at least two .alpha.-helical protein blocks, each having an amino
acid sequence and conformation that allow each .alpha.-helical protein
block to form a coiled-coil with an .alpha.-helical protein block on
another molecule of the block copolymer; and at least one water-soluble,
random-coil protein block, the random-coil protein block linking at least
two of the .alpha.-helical protein blocks.
The random-coil block Y can have the sequence [(AlaGly).sub.p
ProGluGly].sub.n (SEQ ID NO: 23), where p is 0 to 4 (e.g., 1, 2, or 3) and
n is 5 to 100 (e.g., 8 to 54).
The sequences of amino acids that make up X and Z have an (ABCDEFG).sub.m
(SEQ ID NO: 24) pattern, where m is 4 to 100 (e.g., 6 to 18); A and D are
hydrophobic amino acids; E and G are polar amino acids; and B, C, and F
can be any amino acid. In some cases, more than 80% of the E and G amino
acids in X and Z are acidic amino acids. In other cases, more than 80% of
the E and G amino acids in X and Z are basic amino acids. Alternatively,
more than 80% of the E and G amino acids of X can be acidic amino acids
while more than 80% of the E and G amino acids of Z can be basic amino
acids. In another alternative, more than 80% of the E amino acids of X and
G amino acids of Z can be acidic amino acids while more than 80% of the E
amino acids of Z and G amino acids of X can be basic amino acids. In still
another alternative, more than 80% of the E amino acids of X and Z can be
acidic amino acids while more than 80% of the G amino acids of X and Z can
be basic amino acids.
More than 80% of the D amino acids of X and Z can be leucine, for example,
or trifluoroleucine.
The block copolymer can also include linker proteins that link the
.alpha.-helical protein blocks to the random-coil block protein.
In some cases, X and Z are at least 90% identical to each other. X and Z
can be non-identical.
The block copolymer can also include a recognition element that
specifically binds to a cell (e.g., a fibroblast) or to a macromolecule
(i.e., the element binds preferentially to the target cell or molecule in
a sample including the target, but does not bind to other cells or
molecules in the sample). Examples of such recognition elements include
the heparin-binding domain, the endothelial-binding domain, or the
sequence ArgGlyAsp. The recognition element is generally continuous with
the peptide chain that makes up the copolymer, and can be incorporated
either within the random-coil block, between separate blocks (e.g.,
linking the .alpha.-helical block and the random-coil block), or at an end
of the peptide sequence. Alternatively, the recognition element can be
bound to the copolymer via hydrophobic interactions, electrostatic
interactions, disulfide bonds, or hydrogen bonds.
The invention also features a gel that includes a liquid (e.g., an aqueous
liquid such as water) and a block copolymer suspended in the liquid. The
suspension can be monodisperse.
In another aspect, the invention features a method for making a block
copolymer. The method includes the steps of obtaining host cells including
an expression vector having a DNA sequence that encodes the amino acid
sequence of the block copolymer; culturing the host cells under conditions
and for a time sufficient to express the block copolymer; and isolating
the block copolymer from the host cells.
Yet another embodiment of the invention is a method of using a block
copolymer to stimulate regeneration of tissue around a wound. The method
includes the steps of dissolving the copolymer in a liquid to form a
solution, and treating the wound with the solution to form a gelatinous
scaffold for tissue regeneration.
Still another embodiment of the invention is a wound dressing that includes
a block copolymer and an antibiotic compound (e.g., bacitracin, neosporin,
erythromycin), where the copolymer and the antibiotic are both dissolved
in a liquid.
In another aspect, the invention features nucleic acids encoding the new
block copolymers. Examples of suitable nucleic acids can include the
following sequences:
GGT GAC CTG GAA AAC GAA GTG GCC CAG CTG GGA AGG GAA
GTT AGA TCT CTG GAA GAT GAA GCG GCT GAA CTG GAA CAA
AAA GTC TCG AGA CTG AAA AAT GAA ATC GAA GAC CTG AAA
GCC GAA (SEQ ID NO:21); and
GGT GAC CTG AAA AAC AAA GTG GCC CAG CTG AAA AGC AAA
GTT AGA TCT CTG AAA GAT AAA GCG GCT GAA CTG AAA CAA
GAA GTC TCG AGA CTG GAA AAT GAA ATC GAA GAC CTG AAA
GCC AAA (SEQ ID NO:20).
The invention also features a vector that includes this nucleic acid
operatively linked to a promoter. As used herein, the term "operatively
linked" means that selected DNA, e.g., encoding the copolymers, is in
proximity with a promoter, e.g., a tissue-specific promoter, to allow the
promoter to regulate expression of the selected DNA. In addition, the
promoter is located upstream of the selected DNA in terms of the direction
of transcription and translation. Suitable promoters include the P.sub.lac
promoter, the T5 promoter, the adenovirus major late promoter, early and
late promoters of SV40, CMV promoter, TH promoter, RSV promoter, or B19p6
promoter (Shad et al., J. Virol., 58:921, 1986). The promoter may
additionally include enhancers or other regulatory elements.
The invention also features a host cell (e.g., a prokaryote such as E. coli
or other bacteria, or a eukaryote such as a fungus, e.g., yeast).
Unless otherwise defined, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in
the art to which this invention belongs. Although methods and materials
similar or equivalent to those described herein can be used in the
practice or testing of the present invention, suitable methods and
materials are described below. All publications, patents, manufacturers'
technical information, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict, the
present application, including definitions, will control. In addition, the
materials, methods, and examples are illustrative only and not intended to
be limiting.
The new polymers have numerous advantages over many existing gel-forming
polymers. For example, since any given batch of the new polymers can be
produced biologically from a single template, virtually all of the
molecules in that batch will be of equal size; gels formed by
intermolecular binding of the .alpha.-helical blocks are therefore
monodisperse. Monodisperse gels have a uniform pore size that typically
depends on the length of the random-coil block.
Standard molecular biological techniques (e.g., automated DNA synthesis)
allow any amino acid sequence to be encoded by a gene and expressed in
vivo. These techniques enable many characteristics of the new polymers,
and therefore of the new suspensions, to be precisely controlled. Examples
of the characteristics that can be controlled include: the lengths of the
helical and random-coil blocks, the hydrophilicity or hydrophobicity of
any of the blocks, the pore size of the gels formed from the polymer
suspensions, and the responsiveness of the gels to pH and temperature
changes.
Microorganisms can synthesize, in relatively large quantities, high
molecular weight materials that are inherently stereoregular,
monodisperse, and of controlled sequence. Stereoregularity and
monodispersity are rarely achieved by conventional methods of
polymerization such as step, chain, ring-opening, and coordination
methods. Biosynthetic polymers, on the other hand, can exhibit both of
these advantages, and the degree of structural control afforded by
biosynthesis extends to the secondary, tertiary, and quaternary levels.
The physical properties associated with these biomaterials can be
developed on the bases of shape, hydrophilic/hydrophobic character, and
charge placement. Moreover, designing and synthesizing polymeric materials
biosynthetically allows control over the structure of the materials on
both the microscopic and macroscopic levels.
Additionally, a recognition sequence or other peptidic target sequence can
advantageously be inserted into, for example, the random-coil blocks of
the new block copolymers by splicing a gene encoding that sequence into
the template for the new copolymers. The splicing procedure can simply
involve digestion of the template with a restriction enzyme followed by
ligation with the gene encoding the sequence. Gels having an integral
target sequence can be used, for example, in affinity chromatography.
Yet another advantage of the new copolymers is that they have relatively
low molecular weight when compared to many other gel-forming molecules.
Their low molecular weight can result in decreased viscosity of solutions
of the copolymers while still affording high viscosity gels under suitable
conditions.
Other features and advantages of the invention will be apparent from the
following detailed description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B are helical wheel representations of coiled-coil
structures.
FIG. 2 is a general schematic of the physical gelation process of a
monodisperse triblock copolymer.
FIG. 3 is a representation of the amino acid sequences for recombinant
leucine zipper proteins.
FIGS. 4A, 4B, and 4C are helical wheel representations of parallel
coiled-coils of, respectively, a homodimer of an acidic leucine zipper
protein called A1, a homodimer of a basic leucine zipper protein called
B1, and a heterodimer of A1 and B1.
FIGS. 5A, 5B, and 5C are helical wheel representations of antiparallel
coiled-coils of A1 homodimer, B1 homodimer, and A1-B1 heterodimer,
respectively.
FIGS. 6A and 6B are representations of the sequences of coding (5'-3') and
noncoding (3'-5') DNA for linkers L1 and L2, respectively.
FIG. 7 is a schematic diagram of a strategy for cloning artificial genes
encoding random coil and .alpha.-helical blocks into pUC18 plasmid DNA.
FIG. 8 is a representation of the sequence of synthetic DNA encoding
[(AG).sub.3 PEG].sub.10 (SEQ ID NOS: 9,10) inserted into the NruI and SphI
restriction sites.
FIG. 9 is a representation of the sequence of synthetic DNA encoding for
[(AG).sub.3 PEG].sub.28 (SEQ ID NOS: 11,12) inserted into the NruI and
SphI restriction sites.
FIGS. 10A and 10B are representations of the sequence of synthetic DNA
encoding the acidic leucine zipper and the sequence of synthetic DNA
encoding the basic leucine zipper, respectively, inserted into BstEII
restriction sites.
FIG. 11 is a schematic diagram strategy for cloning artificial genes
encoding a helix-coil-helix into pWAP-LIC.sub.10.
FIG. 12 is a schematic diagram of an alternate strategy for cloning
artificial genes encoding a helix-coil-helix into pWAP-LIC2A and
pWAP-LIC2B.
FIG. 13 is a schematic diagram of a strategy for expressing artificial
genes encoding a helix or a coil in Qiagen pQE9 plasmid DNA.
FIG. 14 is a schematic diagram of a strategy for expressing artificial
genes encoding a helix-coil-helix in Qiagen pQE9 plasmid DNA.
FIG. 15 is a plot of the thermal melting profiles of the A1-A1
(.circle-solid.), B1-B1 (.smallcircle.), and A1-B1 (.box-solid.) dimers.
FIG. 16 is a plot of urea denaturation profiles of the A1-A1
(.circle-solid.), B1-B1 (.smallcircle.), and A1-B1 (.box-solid.) dimers.
FIG. 17 is a graph of thermal denaturation curves recorded at 222 nm for A1
homodimer at various pHs.
FIG. 18 is a graph of thermal denaturation curves recorded at 222 nm for B1
homodimer at various pHs.
FIG. 19 is a graph of thermal denaturation curves recorded at 222 nm for
A1-B1 heterodimer at various pHs.
FIGS. 20A, 20B, and 20C are reverse-phase high performance liquid
chromatograms of genetically engineered proteins.
FIG. 21 is a graph of mean square displacement (r.sup.2) as a function of
time at 23.degree. C. to 55.degree. C. for protein L2AC.sub.10 A.
FIG. 22 is a graph of mean square displacement (r.sup.2) as a function of
time for protein L2AC.sub.10 A at 23.degree. C. (a) before and (b) after
thermal cycling.
FIG. 23 is a graph of the concentration dependence of the mean square
displacement as a function of time for protein L2AC.sub.10 A at varying
concentrations.
FIGS. 24A and 24B are graphs of the thermal melting profiles of A1 and B1,
respectively, with Leu (.circle-solid.) and with Tfl (.box-solid.).
DETAILED DESCRIPTION
A new class of block copolymers is disclosed. The new copolymers combine
the properties of coiled-coil leucine zipper proteins with the properties
of flexible, water-soluble, random-coil proteins. Aggregation of the new
copolymers forms three-dimensional, monodisperse gel networks that are
responsive to changes in pH, solvent, and temperature.
Description of the Proteins
The structural complexity of a protein in solution or in the solid state
depends on the chemical nature of both the protein and its environment.
Variables such as pH, temperature, concentration, and chemical structure
can influence the types of conformations that polymers adopt in solution.
Favorable or unfavorable solvent-polymer interactions can also contribute
to the overall conformation of the protein. For example, unfavorable
solvent-polymer interactions can cause protein chains to contract to
exclude solvent, to aggregate, and to precipitate. The correlation between
conformations existing in natural polypeptides and their amino acid
periodicity form the basis for rational materials design.
The block copolymers of the invention have .alpha.-helical blocks,
generally separated by random-coil domains. Other protein blocks (e.g.,
.beta.-sheets and turns) can also be included in the copolymers, bewteen
the .alpha.-helical blocks or the random-coil blocks, or both. The
proteins can be used, for example, to make reversible three-dimensional
gel networks, in which physical aggregates of two or more helical chains
form the junction points of the gel and the random-coil domains control
swelling of the gel. The molecular weight, stereoregularity, sequence, and
function of the new proteins can be concurrently controlled. The block
copolymers with .alpha.-helical and random-coil domains disclosed herein
can form monodisperse gels.
.alpha.-Helical Blocks--Coiled-coils
Certain natural polypeptides (e.g., keratin) can adopt .alpha.-helical
conformations. The axes of individual helices themselves pursue a helical
course to form multistranded cables, or "coiled-coils," probably due to
repeating sequences of amino acids; a seven amino acid repeat, (a b c d e
f g).sub.n (SEQ ID NO: 24), defines a coiled-coil (see FIGS. 1A and 1B).
Proteins having coiled-coil domains are also called "leucine zipper"
proteins, because leucine residues often occur in the a and d positions of
natural helices. In general, a and d are hydrophobic amino acids that
facilitate and stabilize interchain association of .alpha.-helices by
forming complementary hydrophobic helical faces that interact through
hydrophobic and van der Waals interactions.
In addition to the hydrophobic core formed by the a and d residues in
7.sub.2 .alpha.-helices (i.e., having two turns per seven amino acid
residues), the formation of coiled-coils can be further modulated by
interactions between regularly spaced charged groups at specific positions
in the general heptad repeat. Thus, for example, the stabilization of the
coiled-coil helices can be enhanced or diminished in synthetic peptides by
positioning attractive or repulsive charged groups at e and g positions.
Polypeptides can be designed to have the propensity to form parallel or
antiparallel coiled-coils based on charge--charge interactions between the
amino acids in the e and g positions on adjacent helices. In designing
coiled-coil domains, it can be useful to incorporate hydrophilic amino
acids such as Thr, Ser, and Cys at positions b, c, and f to increase
solubility. The seven amino acid units repeat a minimum of four times to
form a 7.sub.2 .alpha.-helical structure.
Parallel and antiparallel arrangements of two leucine zippers are best
illustrated by considering the general positions of residues that are in
proximity to each other. In a coiled-coil structure, positions a and d of
two 7.sub.2 helices are associated at the interface and intertwine to form
a coiled-coil structure with a slight left-handed twist. General
representations of parallel and anti-parallel dimers of two
.alpha.-helical leucine zippers are shown in FIGS. 1A and 1B, wherein one
chain is denoted by a general heptad repeat (a b c d e f g).sub.n (SEQ ID
NO: 24), and the other by (a' b' c' d' e' f' g').sub.n (SEQ ID NO: 24).
FIG. 1A is a representation of a parallel coiled-coil viewed down the
helical axes from the NH.sub.2 -termini of both strands. In the parallel
coiled-coil configuration, the amino acid side chains in the d and a'
positions interact with each other (e.g., those hydrogen bonds or
electrostatic interactions), as do those in the a and d' positions. FIG.
1B is a representation of an antiparallel coiled-coil viewed down the
helical axes from the NH.sub.2 -terminus of the left strand and the
COOH-terminus of the right strand. In the antiparallel arrangement, the
amino acid side chains in the a and a' positions interact with each other,
as do those in the d and d' positions. Interhelical hydrophobic
interactions are present between the a, a', d, and d' groups whereas the
e, e', g, and g' form electrostatic interhelical interactions.
In addition to those .alpha.-helical blocks described in detail above,
there are many other .alpha.-helical blocks that form coiled-coils and
would thus be suitable for use with the present invention. Examples
include natural transcription factor proteins such as Fos, Jun, C/EBP,
CREB, and GCN4; natural muscle and structural proteins such as
tropomyosin, myosin, paramyosin, streptococcal M-protein, and desmoplakin;
and other miscellaneous protein fragments such as S. cerevisiae heat shock
transcription factor (HSF), fibrinogen, laminin, tenascin, macrophage
scavenger receptor protein, bacteriophage leg fiber protein,
.alpha.-actinin, dystrophin, ColE1 Rop protein, tobacco mosaic virus coat
protein, regions of the influenza virus haemagglutinin glycoprotein, and
derivative and analogs of any of these proteins. Still other suitable
examples can be found in Cohen et al., Proteins: Structure, Function, and
Genetics, 7:1 (1990).
In addition to forming dimers, leucine zippers are also known to form
trimeric and tetrameric aggregates. These higher order aggregate states
are referred to as multiple-helix bundles instead of coiled-coils.
Coiled-coils generally contain a narrow hydrophobic surface whereas
multiple helix bundles have a wide hydrophobic surface. The latter surface
involves additional hydrophobic residues in the packing arrangement. The
conformation of the core amino acid side chains determines whether a two-,
three-, or four-stranded oligomerization state will predominate.
The stabilization of coiled-coils can be modulated by both intra- and
interhelical electrostatic interactions. Attractive or repulsive
interactions along the helix affect the stability of an individual
.alpha.-helix as well as the state of association of multiple helices.
Random Coil Blocks
In the present invention, the random coil block has the structure
[(AG).sub.p PEG].sub.n (SEQ ID NO: 23), where p is 0 to 4 and n is 5 to
100. However, nearly any sequence that does not form an .alpha.-helical or
.beta.-sheet structure can be used. Such amino acid sequences do not have
an inherent higher order structure, and are thus described as random coil
blocks in the present context.
Particularly suitable as random coil blocks are sequences that have
ionizable side chains since these side chains are easily hydrated and
afford the desired gelation properties. Nevertheless, it can be desirable
to have few ionizable side chains if the goal is to form gels in less
polar solvents, such as diethyl ether.
Preparation of the Proteins
The production of genetically synthesized materials generally begins with
the insertion of a piece of DNA (e.g., chemically synthesized, or
isolated, or derived from a natural source) into a circular cloning vector
through a series of cutting and ligating reactions. The DNA encodes a
specific sequence of amino acids.
Once the DNA sequence is verified, this piece of DNA is cut from the
cloning vector and inserted into an expression vector or plasmid that
allows for protein production in a prokaryotic or eukaryotic host
microorganism such as a bacterium (e.g., E. coli) or a yeast (e.g., S.
cerevisiae). The microorganism can then be grown, thereby initiating
production of the protein. After an allotted amount of time (i.e., 1 to 5
hours, preferably about 3 hours), the proteins are isolated, purified, and
characterized. If the protein is not secreted by the microorganism, the
cells can be lysed to facilitate protein isolation.
In the new copolymers, .alpha.-helical and random-coil proteins are
combined in a single chain to impart both rigidity, through the helical
segment, and flexibility, through the coiled segment. The general design
of the copolymer can be, for example, a helix-coil-helix motif, XYZ, where
the helices X and Z are leucine zippers. However, other block copolymers
having at least two .alpha.-helical blocks and at least one random-coil
block are also contemplated. For example, a copolymer having repeating
[helix-coil] motif can be suitable. The helical end blocks recognize and
associate with each other through interchain hydrophobic and electrostatic
interactions. The random-coil domains, Y, act as flexible water-soluble
spacers inserted between these ends.
The formation of the helix-coil-helix copolymers as a single chain
generally proceeds as follows. Two separate pieces of DNA are chemically
synthesized to incorporate the sequences encoding the random coil and the
helical blocks, individually (see FIG. 7, for example), each flanked by
restriction sites. By digesting the pieces of DNA with the appropriate
restriction enzymes, then ligating the digestion products, fragments
encoding triblocks (or higher multiblock copolymers) can be assembled. Two
examples of triblock formation are shown in FIGS. 11 and 12. As shown in
FIG. 13, the blocks of the triblock-encoding sequences can be inserted
into an expression vector by digesting with a restriction enzyme that cuts
both at an insertion site in the vector and at the ends of the
triblock-encoding sequence. Multiblock-encoding sequences could similarly
be ligated into the vector (FIG. 14). Examples of multiblock copolymers
can also have a repeated helix-coil motif, as in
helix-coil-helix-coil-helix-coil. Additionally, nucleic acids encoding
other amino acid sequences such as .beta.-sheets, turns, and recognition
sequences that bind specifically to macromolecules or cells can be ligated
into the vector. Moreover, although the nucleic acids encoding the various
blocks of the copolymers can be ligated into a single vector (i.e.,
allowing the entire block copolymer to be expressed as a single,
continuous peptide chain), the individual blocks can alternatively be
encoded by nucleic acids in vectors in separate host cells. In this case,
the blocks can be covalently linked to each other by standard methods
(i.e., after the protein blocks are expressed by the respective host
cells) to form the complete copolymers.
Gelation
The new polymers spontaneously self-organize into well-defined,
supramolecular networks under a given set of conditions (e.g., generally
10-45.degree. C., pH 6-10). The networks can be designed to gel at a given
pH by changing the charge pattern at the e and g positions of the helix.
For example, more acidic side chains in these positions would cause the
networks to gel at lower pH. The terminal helices aggregate to produce a
three-dimensional network, while the flexible intervening random-coil
segment retains solvent and prevents precipitation of the chain. The
result is a gel that is reversibly responsive to both pH and temperature
changes. The reversibility of gelation results from the destabilizing
effects of pH and temperature on the electrostatic and hydrophobic
interactions of the helical ends.
Gels having melting temperatures (T.sub.m) over 37.degree. C. can be of
particular interest, since the use of gels formed from the new proteins as
delivery vehicles in or on the body will require that the gels are stable
at body temperature. The melting temperature depends on the amount of
energy that must be supplied to the polymeric network to disrupt the
non-covalent bonds (e.g., ionic and van der Waals interactions) that
determine the three-dimensional conformation of the proteins and maintain
the integrity of the gel. The energy required depends on the unique amino
acid sequence and coiled-coil design. The protein unfolds at elevated
temperatures, but returns to the low-energy .alpha.-helical folded
arrangement upon cooling, thereby enabling coiled-coils to form again.
If protein chains with many acidic residues and relatively few basic
residues are suspended in a low pH solution, the sidechain acidic groups
can become protonated, resulting in increased stability of the coiled-coil
regions. The proteins would be likely to precipitate from the solution
because highly associated oligomer states of the helical domains can be
formed. The random-coil domain would also have protonated acidic groups
that would cause it to collapse to a hydrophobically packed globule. As
the pH of the solution is increased, deprotonation of the acidic residues
in the random-coil domain can result in swelling of the flexible spacer;
moderate deprotonation of the acidic groups in the helical domain does not
disrupt the helical--helical packing. The protein therefore behaves as a
noncovalent, gelled network. At still higher pH, the acidic residues in
the helical domains also become deprotonated, thus creating repulsive
charge--charge interactions between the helices; as a result, the chains
are free to slip past one another. This proposed gelation process is
depicted in a general schematic in FIG. 2.
An illustrative example is provided by the AC.sub.10 A, AC.sub.10 B, and
BC.sub.10 B block copolymers described below in Example 1. In this
example, A is an acidic .alpha.-helical block, B is a basic
.alpha.-helical block, and C.sub.10 is a random-coil block. AC.sub.10 A
precipitates from aqueous solutions at pH values lower than 6, is a gel
between pH 6 and 9.5, and forms a homogeneous solution at pH values over
9.5. AC.sub.10 B's phase transitions are shifted to higher pH values,
having a gel phase from pH 7 to 10. BC.sub.10 B is a gel only at pH values
above 9. Thus, a correlation is observed: the more basic residues that are
incorporated into the .alpha.-helical blocks, the higher the pH required
for both gelation and dissolution.
Hydrogels belong to a class of functional materials that respond to a
variety of stimuli in aqueous environments. They can be designed to swell
or shrink under certain physiological conditions (e.g., at high or low
salt concentration, pH, or temperature) and they can be used to
encapsulate cells, drugs, and other molecules for site specific use. For
example, since the random coil groups can include charged groups (e.g.,
repeated segments that include glutamic acid residues), the gels can swell
or shrink depending on whether the conditions stabilize or destabilize the
charges. In the example in which the segments include many acidic
residues, increases in the pH would result in an increased number of
deprotonated carboxylic acid residues, thus increasing charge--charge
repulsion and causing the gel to swell. Conversely, decreases in pH would
protonate the carboxylic acid residues and allow the gel to shrink.
Variables such as peptide sequence, stereochemistry, and length can be
precisely controlled by genetic engineering. Each of these variables can
affect the temperature, pH, structural packing, and mechanical properties
of the gels. Proteins having molecular weights of up to about 190,000 or
higher can be made via genetic engineering and can be useful as precursors
of monodisperse gels. Non-covalent protein networks can be made wherein
the strength of the physical crosslinks, the pore size, and the pH and
temperature at which the materials gel are also controlled by varying the
amino acid sequence. For example, by matching acidic residues or a helical
segment of one copolymer (e.g., in the e & g positions of a heptad repeat
unit) with basic residues or helical segment of another copolymer, the
physical crosslinks can be made stronger. By shortening the random coil of
each copolymer, the pore size can be decreased. The statistical end-to-end
distance of a random coil changes with increased molecular weight.
Polymers that Include Non-natural Amino Acids
The incorporation of non-natural amino acids into protein-based materials
provides a route for introducing new chemical functionality and physical
properties into natural polymer systems. Amino acid analogs can be
introduced into cellular proteins either by direct incorporation during
protein synthesis or by post-translational modification. There are at
least three methods for incorporating non-natural amino acids into
proteins: in vivo biological synthesis, in vitro chemical and biological
synthesis, and chemical synthesis by solution or solid-phase methods.
5',5',5'-trifluoroleucine (Tfl), for example, can be incorporated into E.
coli proteins in the absence of its natural analog, leucine (Leu).
Fluorine can mimic the geometry of hydrogen (i.e., the van der Waals radii
of fluorine and hydrogen are 1.35 .ANG. and 1.2 .ANG., respectively),
although they differ in chemical character (e.g., the electronegativity of
fluorine is 3.98 while that of hydrogen is 2.2). Tfl imparts unique
chemical and physiological stability to proteins. For example, the
trifluoromethyl group is lipophilic, making it an ideal candidate for the
modification of membrane spanning or hydrophobic proteins. Additionally,
fluorine-containing amino acids can be studied with nuclear magnetic
resonance (i.e., .sup.19 F-NMR) to monitor structural changes in proteins
or to determine the motion of fluorinated proteins within lipid bilayers.
The incorporation of Tfl into the helical chain at the d position of the
heptad repeat of an .alpha.-helical block, which normally encodes leucine
during protein synthesis, can be accomplished, for example, using a
leucine auxotrophic strain of E. coli. The use of leucine auxotrophs to
prepare unique coiled-coil proteins containing Tfl groups can be applied
to a variety of proteins where Leu participates in folding, stability,
activity, or function. The enhancement of certain physical properties in
natural polymer systems can be achieved for biological or materials
applications.
Other non-natural amino acids can be incorporated into the copolymers by
using the appropriate auxotrophs. For example, selenomethionine,
p-fluorophenylalanine, 3-thienylalanine, azetidine-2-carboxylic acid,
3,4-dehydroproline have been incorporated in E. coli. The last two of
these non-natural amino acids can be used to add chemical functionality to
the copolymers. For example, dehydroproline has a reactive double bond
that can be reacted with oxidizing agents, nucleophiles, or reducing
agents. Furthermore, deuterated amino acids can be incorporated into the
copolymers to afford, for example, sufficient contrast for neutron
scattering studies.
Uses of the Gels
The reversible gelling systems can be used, for example, in the molecular
recognition of macromolecules, as adhesives (e.g., for bonding glass,
metal, or polymers), in thermoreversible and non-covalent fiber networks,
and in recyclable materials. The gels can be used as bases for cosmetics,
for wound management or wound dressings, or to encapsulate drug molecules
for sustained delivery applications.
Because the properties of the gels can be highly sensitive to physical
conditions, the gels can find application in gel-based actuators, valves,
sensors, motors, switches, artificial muscles, memory devices, optical
shutters, filters, toys, paints, coatings, absorbants, bioreactors,
micromachines, display devices, and robotics.
The invention will be further described in the following examples, which do
not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
Synthesis of Genetically Engineered Monodisperse Block Copolymers with
Helix-Coil-Helix Domains
Two similar coiled-coil proteins, A1 and B1, were used as associating
.alpha.-helical blocks. A schematic drawing of the tertiary structure of
these proteins is shown at the top of FIG. 3. Both A1 and B1 proteins
contain different 42-amino acid sequences (six internal heptad repeats, a
b c d e f g) (SEQ ID NOS: 1 and 2, respectively) which constituted the
coiled-coil region of the respective proteins. Each of the 42-amino acid
sequences is flanked by the same 14-amino acid N-terminal chain (SEQ ID
NO: 3) and 18-amino acid C-terminal chain (SEQ ID NO: 4). Residues that
would increase the solubility of the coiled-coil complex in water (e.g.,
glutamic acid, glutamine, aspartic acid, asparagine, arginine, and serine)
were chosen for the b, c, and f positions. Acidic and basic groups,
glutamic acid and lysine, respectively, were placed at the e and g
positions to provide intermolecular attraction or repulsion between
chains. The placement of oppositely charged groups at the e and g
positions in the A1 and B1 proteins was designed to maximize heterodimer
formation over either homodimer when solutions equimolar in A1 and B1 were
mixed. The underlined regions of the A1 and B1 Helix segments in FIG. 3
emphasize this charge pattern at positions e and g. Helical wheels
representing parallel and antiparallel arrangements of A1 and B1
homodimers and heterodimers are shown in FIGS. 4 and 5, respectively. The
charge pattern at the e and g positions along the heptad repeat
distinguishes A1 from B1; the polar charged groups at these positions
determine whether the protein chain is predominantly acidic or basic.
The physical properties of the A1 and B1 complexes are listed in Table 1.
In the columns labelled "interactions" in the table, A indicates an
attractive (i.e., acid-base) interaction and R indicates a repulsive
(i.e., acid-acid or base-base) interaction. The calculated interactions
include e-g' and e'-g for parallel interhelical dimer interaction, and
e'-e and g-g' for antiparallel interhelical dimer interaction. Intrachain
interactions include b-f (i to i+4), b-e (i to i+3), c-g (i to i+4), e-b
(i to i+4), f-c (i to i+4), f-b (i to i+3) and g-c (i to i+3).
Dimer-tetramer dissociation constants were measured by analytical
ultracentrifugation for protein solutions (24 to 220 .mu.M). 95%
confidence limits are shown in parentheses.
TABLE 1
______________________________________
Parallel Dimer-
Charge Interchain
Antiparallel
Intrachain
Tetramer
Pattern Inter- Interchain
Inter- K.sub.d
Protein
at e/g actions Interactions
actions
(in .mu.M)
______________________________________
A1 EEEEEE 4A 6R 2A 8R 12A 16R
76
EKKEKE (50-112)
B1 KKKKKK 6A 4R 4A 6R 12A 16R
123
.sup. KEEEKK (1-6)
A1-B1 -- 5A 5R 7A 3R 12A 16R
--
______________________________________
Synthesis and Purification of Single-Stranded DNA
Oligonucleotides encoding proteins A1, B1, and random-coil domains were
synthesized on a Biosearch Model 8700 DNA synthesizer by phosphoramidite
chemistry, and cleaved from polymer supports with 30% ammonium hydroxide
at 65.degree. C. The polymer supports were removed by centrifugation and
the decanted supernatant was dried in a speed vacuum. The remaining
pellets were then resuspended in 1 ml of deionized distilled water
(ddH.sub.2 O) and centrifuged at 15,800 g for 10 minutes at 25.degree. C.
to remove any insoluble materials. The remaining aqueous solution of DNA
was adjusted to a final concentration of 10 mM magnesium chloride.
Absolute ethanol (equilibrated at 25.degree. C.) was added to the DNA
solution in a 3:1 ratio.
The mixtures were then cooled at -70.degree. C. for 10 minutes and pelleted
at 15,800 g for 10 minutes at 4.degree. C. The supernatant was decanted
and the pellet dried in a speed vacuum. The resulting pellets were
resuspended in 1 ml of ddH.sub.2 O and single-stranded DNA was quantitated
based on optical absorbance measurements at 260 nm.
Polyacrylamide gel electrophoresis (PAGE; 10% polyacrylamide, 8 M urea) was
used to purify the single-stranded DNA. FIGS. 6A and 6B are the sequences
of linkers L1 and L2, respectively, showing both the coding (SEQ ID
NOS:5,7) and noncoding (SEQ ID NOS: 6,8) strands incorporated in the
HindIII and EcoRI restriction sites of cloning vector pUC-18. Important
restriction sites are underlined and .quadrature. indicates a deletion of
a nucleotide. These strands were mixed with 2.times. formamide loading
buffer (containing 0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30%
glycerol in water) and heated at 95.degree. C. for 5 minutes before
loading onto the preheated gel. The samples were electrophoresed at
constant voltage (350 V) for approximately 2 hours. The purified bands
corresponding to the purified DNA strands were visualized by ethidium
bromide and excised from the gel. Gel slices were crushed in 1 ml of
elution buffer (containing 500 mM ammonium acetate; 0.1% sodium dodecyl
sulfate, SDS; 10 mM magnesium acetate, and 1 mM ethylenediaminetetraacetic
acid dipotassium salt dihydrate, pH 8.0), and incubated overnight at
37.degree. C. on a spin wheel.
Gel residue was removed by centrifugation at 4.degree. C. in filter vials
and the supernatants were extracted with 1-butanol to remove ethidium
bromide, and then treated with 10 .mu.l of Type II oyster glycogen (10
.mu.g/ml), sodium chloride (250 mM), and 0.9 ml absolute ethanol. Mixtures
were precipitated at -20.degree. C. overnight, centrifuged at 4.degree. C.
for 30 minutes, then decanted. The DNA pellets were washed once with 500
.mu.l of cold 70% ethanol and redissolved in 50 .mu.l of ddH.sub.2 O.
Phosphorylation and Annealing of Single-Stranded DNA
The purified oligonucleotides (L1: coding=5 .mu.g, noncoding=5 .mu.g and
L2: coding=5 .mu.g, noncoding=5 .mu.g) were suspended in about 25 .mu.l of
ddH.sub.2 O, 5 .mu.l of 10.times. T4 polynucleotide kinase buffer
(containing 80 mM Tris-HCl pH 7.5, 20 mM dithiothreitol, DTT, 10 mM
magnesium chloride, and 1 mM adenosine triphosphate, ATP), 2 .mu.l of 100
mM ATP, and 1 .mu.l of T4 polynucleotide kinase (10 Richardson units), to
make a final volume of 50 .mu.l. The reaction tubes were then incubated at
37.degree. C. for 45 minutes and 1 .mu.l of sodium chloride (5 M) was
added to each vial for a final sodium chloride concentration of 100 mM.
The mixtures were placed in a 95.degree. C. water bath and equilibrated to
room temperature over 10 hours. The annealed duplexes were then extracted
with 50:50 phenol:chloroform and washed once with 100% chloroform. 100
.mu.l of isopropanol and 20 .mu.l of 3 M sodium acetate were added to each
vial and the vials were placed in the -70.degree. C. freezer for 30
minutes. The DNA duplexes were pelleted at 4.degree. C. (15,800 g) for 25
minutes and then dried in the speed vacuum. The pellets were resuspended
in 100 .mu.l of ddH.sub.2 O.
Ligation of L1 and L2 Duplexes into pUC-18
2.5 .mu.g of Pharmacia pUC-18 cloning vector was digested with EcoRI (2
units where a unit is the amount of enzyme required to completely digest 1
.mu.g of substrate DNA in a total reaction volume of 50 .mu.l) and HindIII
(2 units) restriction enzymes. The DNA was visualized on a 1% agarose gel
with ethidium bromide and the linearized pUC-18 band was cut and purified
with a BIO-RAD.TM. gel extraction kit. A 33 molar excess of linker to
vector (equal volumes) was combined with 2 .mu.l of 10.times. ligase
buffer (50 mM Tris-HCl, pH 7.8; 10 mM magnesium chloride; 10 mM DTT; 1 mM
ATP; and 25 .mu.g/ml bovine serum albumin, BSA), 0.5 .mu.l T4 ligase (15
Weiss units), and 7.6 .mu.l of water to make a total volume of 20 .mu.l.
The resulting ligation mixtures were placed in a 15.degree. C.
refrigerator for 21 hours.
Transformation of pUC-18 Containing L1 and L2 into DH5.alpha.F' Competent
Cells
DH5.alpha.F' cells were grown to an optical density at 550 nm (OD.sub.550)
of 0.573, placed on ice for 10 minutes, and collected by centrifugation at
2000 g for 15 minutes at 4.degree. C. The cells were then resuspended in 2
ml of TFB1 Buffer (10 mM morpholinoethanesulfonic acid (MES) pH 6.2; 100
mM rubidium chloride, 10 mM calcium chloride dihydrate, and 50 mM
manganese chloride tetrahydrate) by gentle tilting of the tube, adjusted
to a final volume of 16 ml. This suspension was left on ice for 15 minutes
before the cells were pelleted at 2000 g for 15 minutes and then
resuspended in TFB2 buffer (10 mM 3-(N-morpholino)propanesulfonic acid,
MOPS; 75 mM calcium chloride dihydrate; 10 mM rubidium chloride; and 15%
glycerol). The tube was left on ice for 15 minutes before 200 .mu.l
aliquots were dispensed into vials and then stored at -70.degree. C.
Later, competent cells were thawed on ice and 50 .mu.l of the cells were
added to 10 .mu.l of the ligation mixtures. The vials were then placed on
ice for 2 hours and heat shocked for 3 minutes at 42.degree. C. The vials
were then placed on ice for 5 minutes. 500 .mu.l 2xYT medium containing 16
g casein hydrolysate, 10 g yeast extract, and 5 g sodium chloride per
liter) was added to the mixtures and incubated for 45 minutes at
37.degree. C. The cells were then spread onto agar plates containing 200
.mu.g/ml ampicillin, and grown for 17 hours.
Mini-preps of pUC-18 Containing L1 and L2 Transformants
pUC-18 cloning plasmids containing L1 and L2 DNA were designated as pWAP-L1
and pWAP-L2 respectively (see FIG. 7). Eight colonies of pWAP-L1 and
twelve colonies of pWAP-L2 were grown overnight in 2xYT medium containing
ampicillin (200 .mu.g/ml).
The DNA from the cell cultures was purified by the following miniprep
protocol. A 1.5 ml saturated culture was spun at 13,600 g for 2 minutes
and the supernatant was poured off. The cells were resuspended in 200
.mu.l of GTE buffer (containing 50 mM glucose; 25 mM Tris-HCl, pH 8; and 1
mM EDTA, pH 8.0) by repeated pipetting. 200 .mu.l of 3 M sodium acetate
(pH 4.8) was added to neutralize the solution, and the mixture was
incubated on ice for 5 minutes. The resulting white precipitate was
collected by centrifugation at 13,600 g for 5 minutes. The clarified
supernatant was transferred to a new 1.5 ml tube containing 2 .mu.l of
RNAse A (10 mg/ml) and incubated at 50.degree. C. for 30 minutes. 500
.mu.l of 50:50 phenol:chloroform was added, and the mixture was spun at
13,600 g for 5 minutes. The aqueous layer was decanted and washed once
with chloroform. 500 .mu.l of isopropyl alcohol was added. DNA was
precipitated for 30 minutes at -20.degree. C. and pelleted at 13,600 g for
30 minutes at 4.degree. C. The supernatant was discarded and the DNA was
resuspended into 100 .mu.l of ddH.sub.2 O before 50 .mu.l of 25%
polyethylene glycol (PEG; 8000 molecular weight) in 2.5 M sodium chloride
was added. This mixture was incubated for 30 minutes at -20.degree. C.,
centrifuged at 13,600 g for 30 minutes, and resuspended in 50 .mu.l of
ddH.sub.2 O.
Screening for Inserts L1 and L2 by Enzymatic Digestions
To verify the proper insertion of L1 and L2 DNA into pUC-18, restriction
digests of pWAP-L1 and pWAP-L2 were carried out by mixing 15 .mu.l of the
plasmid DNA fragments collected in the procedure described above, 5 .mu.l
of NEB2 buffer (containing 50 mM sodium chloride, 10 mM Tris-HCl, 10 mM
magnesium chloride, and 1 mM DTT, pH 7.9), 1 .mu.l HindIII (20 units), 1
.mu.l NheI (5 units), and 33 .mu.l of ddH.sub.2 O to give a final volume
of 50 .mu.l. The resulting mixtures were then heated for 4 hours at
37.degree. C. The digested DNA was visualized by ethidium bromide on a 1%
agarose gel, and colonies confirmed to have the proper inserts were saved
for DNA sequencing. In addition to this digestion, individual digestions
were carried out for pWAP-L1 and pWAP-L2 as further checks for proper
insertion. pWAP-L1 (7 .mu.l) was digested with 0.5 .mu.l of NruI (4
units), 2.5 .mu.l NEBuffer NruI containing 100 mM potassium chloride, 50
mM Tris-HCl, and 10 mM magnesium chloride, pH 7.7), and 15 .mu.l of
ddH.sub.2 O for a final volume of 25 .mu.l. pWAP-L2 was digested with 0.5
.mu.l of XhoI (10 units), 2.5 .mu.l of NEB2 buffer, and 15 .mu.l of
ddH.sub.2 O. The digested DNA was visualized on a 1% agarose gel with
ethidium bromide; pUC-18 was used as a positive control since it contains
neither NruI nor XhoI restriction sites.
DNA Sequencing of pWAP-L1 and pWAP-L2 Inserted into pUC-18 Cloning Vector
The DNA sequences of pWAP-L1 and pWAP-L2 were confirmed by the Sanger
dideoxy sequencing method using the USB SEQUENASE VERSION 2.0.1.TM.
sequencing protocol. Universal (M13/pUC Sequencing Primer (-20)17mer) and
reverse (M13/pUC Sequencing Primer (-47)24mer) were used to read the
coding and anticoding DNA strands, respectively.
Digestion and Removal of Phosphate Groups of pWAP-L1 and pWAP-L2
pWAP-L1 (88 .mu.l, or approximately 220 ng) was first digested with 2.0
.mu.l of NruI (50.8 units) and 10 .mu.l of NruI buffer overnight at
37.degree. C. The DNA was precipitated and isolated, then digested with
1.0 .mu.l of SphI enzyme (5 units), 5 .mu.l of NEB2 buffer, and 44 .mu.l
of ddH.sub.2 O over 5 hours at 37.degree. C. pWAP-L2 (88 .mu.l, or
approximately 1 .mu.g) was digested with 2.0 .mu.l BstEII (20 units) and
10 .mu.l NEB3 buffer (containing 100 mM sodium chloride, 50 mM Tris-HCl,
10 mM magnesium chloride, and 1 mM DDT, pH 7.9) at 60.degree. C.
overnight. Both pWAP-L1 and pWAP-L2 mixtures were heat inactivated for 20
minutes at 65.degree. C., then 1 .mu.l of calf intestinal alkaline
phosphatase (CIP; 10 units) was added to the pWAP-L1 digestion mixture,
and 8 .mu.l of CIP (80 units) was added to the pWAP-L2 digestion mixture.
EDTA was added to each solution to give a final concentration of 5 mM, and
the reaction mixtures were heated to 65.degree. C. for 1 hour. The DNA was
extracted with phenol and precipitated with 20 .mu.l of sodium acetate and
120 .mu.l of 100% ethanol. The precipitated DNA was resuspended in 40
.mu.l of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0) before being run
on a 1% agarose gel. Gel slices were excised, and the DNA was extracted
and purified by Bio-Rad DNA purification method.
Insertion and Verification of Random-Coil and Helical DNA Sequences into
Linearized pWAP-L1 and pWAP-L2
DNA fragments encoding [(AG).sub.3 PEG].sub.10 (FIG. 8; SEQ ID NOS:9,10)
and [(AG).sub.3 PEG].sub.28 (FIG. 9; SEQ ID NOS: 11,12) were ligated into
linearized pWAP-L1 at SphI/NruI. The SphI/NruI sites in the DNA fragments
were derived from a fragment obtained from Protein Polymer Technologies,
Inc (San Diego, Calif.). In addition, DNA fragments encoding
.alpha.-helical acidic leucine zipper (FIG. 10A; SEQ ID NOS: 13,14) and
basic leucine zipper (FIG. 10B; SEQ ID NOS: 15,16) sequences were ligated
into linearized pWAP-L2 at the BstEII site. The restriction sites are
underlined in FIGS. 8, 9, 10A, and 10B. Prior to ligation, the DNA
fragments encoding [(AG).sub.3 PEG] .sub.10 and [(AG).sub.3 PEG].sub.28
were obtained from pET3-5 and pET3-14 plasmids, respectively, by cutting
with SphI and NruI. Likewise, the DNA molecules encoding the acidic and
basic leucine zippers were digested from pUC-LINKA1 with BstEII enzyme.
Purified pieces were ligated into the linear pWAP-L1 or pWAP-L2 by
combining 10 .mu.l of insert, 10 .mu.l of vector, 2.5 .mu.l of ligase
buffer, 0.5 .mu.l of T4 DNA ligase, and 2.0 .mu.l of ddH.sub.2 O.
The ligation mixtures were then used to transform DH5.alpha.F' cells and
the resultant transformants were screened for proper DNA insertion by
enzymatic digestions. Plasmids containing the DNA encoding [(AG).sub.3
PEG].sub.10 and [(AG).sub.3 PEG].sub.28 were digested with NdeI and SphI,
whereas plasmids containing the DNA encoding the acidic and basic leucine
zippers were digested with NdeI and BglII. The sequences of all of the
combined DNA molecules were confirmed by the Sanger dideoxy sequencing
method mentioned previously. Plasmids were designated pWAP-L1C.sub.10,
pWAP-L1C.sub.28, pWAP-L2A, and pWAP-L2B as shown in FIG. 7. One difference
between the L1 and L2 designation is that L1 codes for a single tryptophan
at the C-terminus whereas L2 codes for a single cysteine at this end. A
and B are acidic and basic leucine zipper proteins, respectively, and
C.sub.n is the random-coil [(AG).sub.3 PEG].sub.n (SEQ ID NO: 23) repeat.
The sequences of L2-A, L2-B, and L1-C.sub.10 are shown in Table 2, in
which the A, B, and C.sub.10 sequences are printed in bold-face type.
TABLE 2
__________________________________________________________________________
L2-A:
MRGSHHHHHHGSDDDDKWASGDLENEVAQLEREVRSLEDEAAELEQKVSRLKNEIEDLKA
(SEQ ID NO:17)
EIGDHVAPRDTSMGGC
L2-B:
MRGSHHHHHHGSDDDDKWASGDLKNKVAQLKRKVRSLKDKAAELKQEVSRLKNEIEDLKA
(SEQ ID NO:18)
KIGDHVAPRDTSMGGC
L1-C.sub.10 :
MRGSHHHHHHGSDDDDKASYRDPMGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPE
(SEQ ID NO:19)
GAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGARMPT
SW
__________________________________________________________________________
Enzymatic Digestions of DNA at NheI and SpeI Restriction Sites
To recombine the DNA described above to form DNA that encodes the triblocks
A-C.sub.n -A, A-C.sub.n -B, and B-C.sub.n -B, a series of digestions and
ligations were carried out at the NheI and SpeI enzymatic restriction
sites. FIGS. 11 and 12 represent two different routes to prepare the
coding sequences of the triblock copolymers. The combination of DNA was
achieved through NheI and SpeI sites located outside of the helix and coil
regions of the DNA. NheI and SpeI sites are compatible; upon ligation of a
NheI to a SpeI end, or vice versa, the site is destroyed. Both restriction
sites have the same middle four nucleotide bases that allow them to
combine upon ligation, as shown below:
______________________________________
NheI 5' G C T A G C 3'
3' C G A T C G 5'
SpeI 5' A C T A G T 3'
3' T G A T C A 5'
______________________________________
The frequency at which NheI cut ends combine with SpeI cut ends is
approximately 50%. In all cases, NheI and SpeI digestions were carried out
in a total volume of 25 .mu.l; enzymatic buffer, enzyme, ddH.sub.2 O, and
DNA volumes were adjusted accordingly. Miniprep, transformation, and
phosphate removal procedures were carried out as described above. 2%
Agarose gels were used in the verification, quantification, and isolation
of the various DNA.
DNA encoding twelve different proteins (i.e., A-Cys, B-Cys, C.sub.10 -Trp,
AC.sub.10 A-Trp, AC.sub.10 B-Trp, BC.sub.10 B-Trp, AC.sub.10 A-Cys,
AC.sub.10 B-Cys, BC.sub.10 -Cys, AC.sub.28 A-Cys, AC.sub.28 B-Cys, and
BC.sub.28 B-Cys) was made through the series of steps described above. The
DNA molecules were designated pWAP-L2A, pWAP-L2B, pWAP-L1C.sub.10,
pWAP-L1AC.sub.10 A, pWAP-L1AC.sub.10 B, pWAP-L1BC.sub.10 B,
pWAP-L2AC.sub.10 A, pWAP-L2AC.sub.10 B, pWAP-L2BC.sub.10 B,
pWAP-L2AC.sub.28 A, pWAP-L2AC.sub.28 B, and pWAP-L2BC.sub.28 B,
respectively.
BamHI Digestions of DNA Molecules
pWAP-L2A, pWAP-L2B, pWAP-L1C.sub.10, pWAP-L1AC.sub.10 A, pWAP-L1AC.sub.10
B, pWAP-L1BC.sub.10 B, pWAP-L2AC.sub.10 A, pWAP-L2AC.sub.10 B,
pWAP-L2BC.sub.10 B, pWAP-L2AC.sub.28 A, pWAP-L2AC.sub.28 B, and
pWAP-L2BC.sub.28 B DNA were grown in DH5.alpha.F' host cells in 100 ml
2xYT medium with ampicillin (200 .mu.g/ml) and isolated using Qiagen
maxiprep DNA columns. 43 .mu.l (approximately 300 ng) of each plasmid was
combined with 5 .mu.l of NEB BamHI buffer containing 150 mM sodium
chloride, 10 mM magnesium chloride, 1 mM DDT, pH 7.9 and 2 .mu.l of BamHI
(40 units) and incubated overnight at 37.degree. C. (FIGS. 13 and 14)
Qiagen pQE9 expression vector (Chatsworth, Calif.) DNA was also cut with
BamHI and dephosphorylated by the CIP protocol as previously described
(FIGS. 13 and 14). The fragments were visualized on 2% agarose gels and
excised for purification by BIO-RAD.TM. DNA binding beads. Quantification
of the recovered DNA was made on 2% agarose gels.
Ligation of DNA Molecules
A 5 molar excess of linker to vector was combined with 2.5 .mu.l ligation
buffer, 0.5 .mu.l ligase, and ddH.sub.2 O for a total volume of 25 .mu.l.
The ligated mixtures were transformed into K-12 E. coli strain SG13009
(Qiagen, Chatsworth, Calif.) containing repressor plasmid, pREP4 (Qiagen,
Chatsworth, Calif.). Mixtures were spread onto 2xYT agar plates containing
200 .mu.g/ml ampicillin and 50 .mu.g/ml kanamycin, and incubated for 16
hours at 37.degree. C. Single colonies were grown to saturation in 5 ml of
2xYT medium and the DNA was isolated. Restriction digests were used to
verify orientation, size, and digest sites of the DNA from the transformed
colonies. These were accomplished with NheI, EcoRI/HindIII, and BamHI
restriction enzymes, respectively. Colonies containing the correct inserts
were then used in the following bacterial expression procedures.
Protein Expression in pQE9-derived Expression Vectors
Recombinant vectors (pQE9) containing the targeted sequences were used for
protein expression in host cells of strain SG13009 containing repressor
plasmid (pREP4) (FIGS. 13 and 14). New plasmid designations are pQE9-L2A,
pQE9-L2B, pQE9-L1C.sub.10, pQE9-L1AC.sub.10 A, pQE9-L1AC.sub.10 B,
pQE9-L1BC.sub.10 B, pQE9-L2AC.sub.10 A, pQE9-L2AC.sub.10 B,
pQE9-L2BC.sub.10 B, pQE9-L2AC.sub.28 A, pQE9-L2AC.sub.28 B, and
pQE9-L2BC.sub.28 B. Saturated cultures containing the targeted DNA
sequences were grown in 25 ml of 2xYT medium with ampicillin (200
.mu.g/ml) and kanamycin (50 .mu.g/ml) for 15 hours at 37.degree. C. These
cultures were then transferred to 2 l flasks, each containing 1 l
sterilized 2xYT medium (with ampicillin and kanamycin). The cultures were
incubated for an additional 6 hours at 37.degree. C. with vigorous
aeration until the optical densities at 600 nm (OD.sub.600) were in excess
of 2.1 mM. Isopropyl-.beta.-thiogalactoside (IPTG) was added and protein
synthesis was induced for a period of 4 hours at 37.degree. C. The cells
were then centrifuged (22,100 g for 30 minutes), the supernatant was
removed, and 50 ml of 6 M guanidine-HCl buffer containing dibasic sodium
phosphate (0.1 M, pH 8) was added. The cells were lysed by storing in a
-80.degree. C. freezer, then the supernatant was collected for protein
purification by centrifugation at 22,100 g for 50 minutes. The clarified
supernatant was loaded onto a nickel-NTA affinity column at pH 8 and the
bound target proteins were washed with 8 M urea buffers at pH 8, 7, and 6,
then eluted at pH 5. The appropriate fractions were dialyzed against
deionized water, then they were freeze-dried. The final weights of the
purified target proteins were in the range of 20-160 mg per 1 l of cell
growth medium, after purification with Ni.sup.2+ metal affinity
chromatography.
Matrix Assisted Laser Desorption Mass Spectrometry (MALDI-MS) with
Time-of-Flight Analyzer (TOF)
MALDI-TOF mass spectrometry of the gas-phase protein ions was performed on
a LASERMAT 2000.TM. (Finnigan Mat, San Jose, Calif.) by the Analytical
Chemistry and Peptide/DNA Synthesis Facility at Cornell University,
Ithaca, N.Y. The protein samples were mixed with a molar excess of matrix
solution (0.05 M 3,5-dimethoxy-4-hydroxycinnamic acid for predicted masses
greater than 20,000 and 0.05 M .alpha.-cyano-4-hydroxycinnamic acid for
predicted masses less than 20,000 in 30% acetonitrile/water, 0.1%
trifluoroacetic acid) before approximately 0.5 .mu.l was dried onto the
sample probe. The surface of the probe was irradiated with a nitrogen
laser at 337 nm. Detection of the protein ions were recorded in the
positive ion mode with laser power ranging between 10-25 kV. The results
of the MALDI experiments are summarized in Table 3. The weights are
average molecular weights and refer to the protonated form of the
polypeptides.
TABLE 3
______________________________________
Domain [M + H] + [M + H] +
Relative
Structure (calculated) (observed)
Error (%)
______________________________________
A-Cys 8550 8548 0.02
B-Cys 8545 8559 0.16
C-Trp 10384 10433 0.47
AC.sub.10 A-Trp
22092 22317 1.02
AC.sub.10 B-Trp
22086 22133 0.21
BC.sub.10 B-Trp
22080 22165 0.38
AC.sub.10 A-Cys
22440 22478 0.17
AC.sub.10 B-Cys
22434 22506 0.32
BC.sub.10 B-Cys
22429 22402 0.12
AC.sub.28 A-Cys
34451 33148 3.78
AC.sub.28 B-Cys
34446 34567 0.35
BC.sub.28 B-Cys
34440 35712 3.69
______________________________________
Quantitative Amino Acid Compositional Analysis
Amino acid analysis was performed by the Analytical Chemistry and
Peptide/DNA Synthesis Facility at Cornell University, Ithaca, N.Y. Amino
acid analysis was carried out on a PICO-TAG.TM. Amino Acid Analysis System
(Millipore Corp., Bedford, Mass.). Duplicate samples were hydrolyzed in
constant boiling hydrochloric acid for 110 minutes at 150.degree. C.
Appropriate blanks, controls and standards were hydrolyzed in the same
vessel as the batch hydrolysis. The amino acids were derivatized with
phenyl isothiocyanate (PITC) and analyzed by reversed phase HPLC. The
resulting phenylthiocarbamoyl amino acid derivatives were separated on a
4.6.times.300 mm NOVA PACK.TM. C18 column employing a modified Pico-Tag
buffer system and detected at 254 nm.
The purified proteins were resolved on a polyacrylamide minigel and
visualized by staining with Coomassie Brilliant Blue R-250. The results of
the analysis were all consistent with the desired sequences, within the
error range of the instrumentation.
Thermodynamic Analysis
Thermal and urea denaturation studies were done with 3 .mu.M solutions of
A1, B1, and A1-B1 complexes and the results are shown in FIGS. 15
(thermal) and 16 (urea denaturation). The fraction of unfolded peptide in
FIG. 15 was determined by monitoring CD spectra at 222 nm (3 .mu.M protein
in 50 mM monobasic sodium phosphate, 100 mM sodium chloride, pH 7.4). The
graph in FIG. 15 suggests that the folded-to-unfolded transition
temperature for equimolar mixtures of A1 and B1 is higher than that of
either A1-A1 or B1-B1 solutions. This implies that the A1-B1 coiled-coils
are more stable than A1-A1 or B1-B1 coiled coils. The fraction of unfolded
peptide in FIG. 16 was determined by monitoring CD spectra at 222 nm as a
function of urea concentration over the range of 0 to 9.5 M, with 3 mM
protein concentration in 50 mM monobasic sodium phosphate and 100 mM
sodium chloride, pH 7.4.
The temperature curves showed that the thermal transition temperature,
T.sub.m, from a folded to an unfolded state was higher for the A1-B1
mixture than either of the A1-A1 or B1-B1 solutions. These results were
consistent with urea denaturation profiles that show a higher
[urea].sub.1/2 (transition where 50% of the helix is unfolded at
20.degree. C.) for the unfolding process of A1-B1 than for the A1-A1 and
B1-B1. Thus, at pH 7.4 the heterodimer complexes were more stable than
either of the homodimer complexes.
Table 4 compares the data collected and calculated from these curves for
A1-A1, B1-B1, and A1-B1 interactions. In Table 4, T.sub.M is the
temperature at which 50% of the helix is unfolded in 50 mm sodium
phosphate, 100 mm sodium chloride, ph 7.4; [urea].sub.1/2 is the
concentration of urea at which 50% of the helix is unfolded at 20.degree.
C.; and .DELTA.G.sub.u is the molar free energy of folding calculated from
the urea denaturation profiles. These data showed that the transition
temperature for the heterodimer complex was higher than that of either
homodimer solution.
TABLE 4
______________________________________
.DELTA.G.sub.u
.DELTA..DELTA.G.sub.u
Dimer T.sub.m in PBS
[Urea].sub.1/2
slope (Kcal/
(Kcal/
Complex (.degree. C.)
(M) m mole) mole)
______________________________________
A1--A1 58.3 3.57 -1.295 11.54 0.00
B1--B1 75.5 3.66 -1.512 12.55 0.13
A1-B1 86.3 5.76 -1.165 13.76 2.75
______________________________________
A two state mechanism was used to describe the equilibrium between folded
and unfolded protein states. Table 4 indicates that the free energy of
unfolding, .DELTA.G.sub.u, and the difference in the free energies of
unfolding, .DELTA..DELTA.G.sub.u, also increase upon mixing of equimolar
solutions of A1 and B1. .DELTA.G.sub.u is obtained by plotting lnK.sub.d,u
(i.e., the equilibrium constant for dissociation of the dimers) against
the concentration of denaturant, then extrapolating back to zero
denaturant concentration. .DELTA..DELTA.G.sub.u is determined by comparing
the free energy values of proteins that are calculated when 50% of the
protein has unfolded in the presence of a denaturant. The latter method
minimizes any errors introduced in the measurement of the slope by
extrapolation to zero.
Thermal denaturation curves as a function of pH were recorded at 222 nm
with 12 .mu.M solutions of A1, B1, and the A1-B1 mixture (in 10 mM
monobasic sodium phosphate at pH 2.1, 7.1, 8.1, and 10.1) and are shown in
FIGS. 17, 18, and 19, respectively. At lower pH values, the thermal
transition occurred at higher temperatures for A1. It can be theorized
that this is because the A1 chains contain carboxylic acid groups at the e
and g positions in the heptad repeat and lower pH values can therefore
promote the formation of coiled coils as the sidechains remain protonated.
At higher pH values, the negatively charged carboxylate groups can cause
interchain electrostatic repulsion and reduce the stability of the coiled
coil structure. The opposite trend was observed for B1, consistent with
the argument above; the B1 chains have basic lysine sidechains at the e
and g positions. The data for the equimolar mixture of A1 and B1 showed
that at every pH the transition shifted to a higher temperature.
Example 2
Reversible Hydrogels from Self-Assembling Artificial Proteins
Oligonucleotides were synthesized on a BIOSEARCH.TM. Model 8700 DNA
synthesizer and ligated into the polylinker region of the pUC18 cloning
vector. A 213 bp fragment encoding .alpha.-helical protein A-Cys was
ligated into the EcoRI and HindIII restriction sites of pUC18 to yield
pWAP-L2A. Likewise, a 351 bp fragment encoding random-coil protein
C.sub.10 -Trp was ligated into the EcoRI and HindIII restriction sites of
pUC18 to yield pWAP-L1C. Recombinant DNA was cloned in E. Coli strain
DH5.alpha.F' before coding and non-coding sequences were verified by DNA
sequence analysis. DNA fragments that encode A-Cys and C.sub.10 -Trp
independently were used to form recombinant DNA that encodes AC.sub.10
A-Cys. This combination of DNA was achieved through NheI and SpeI sites.
All DNA fragments were isolated by BamHI digestion, and directionally
ligated into the E. coli expression vector pQE9 (Qiagen, Chatsworth,
Calif.) to form an NH.sub.2 -terminal His fusion product. Recombinant DNA
plasmids encoding A-Cys, C.sub.10 -Trp, and AC.sub.10 A-Cys were
designated pQE9-L2A, pQE9-L1C.sub.10, and pQE9-L2AC.sub.10 A,
respectively. The host used for protein expression was E. coli strain
SG13009, containing the pREP4 repressor plasmid (Qiagen, Chatsworth,
Calif.). Cultures containing these DNA sequences were grown at 37.degree.
C. in 2 l of TB medium (containing 16 g Bacto-Tryptone, 10 g yeast
extract, and 5 g sodium chloride per liter) with ampicillin (100 .mu.g/ml)
and kanamycin (50 .mu.g/ml) until the optical densities at 600 nm
(OD.sub.600) were in excess of two. 1 mM of
isopropyl-.beta.-thiogalactoside (IPTG) was added and protein synthesis
was induced for a period of 4 hours at 37.degree. C. The cells were then
centrifuged at 22,100 g for 30 minutes, and 50 ml 6 M guanidine-HCl buffer
containing dibasic sodium phosphate (0.1 M, pH 8) was added. The cells
were lysed by storing in a -80.degree. C. freezer and the supernatant was
collected for protein purification. Metal affinity chromatography was used
to purify the target proteins, using nickel(II)nitrilotriacetic acid
(Qiagen, Chatsworth, Calif.). Purified protein yields of A-Cys, C.sub.10
-Trp, and AC.sub.10 A-Cys were 122 mg, 26 mg, and 56 mg per liter of
growth medium, respectively.
Additional Purification and Verification of Protein Synthesis
Reverse-phase high performance liquid chromatographs (RP-HPLCs) were
acquired on a Waters system (including a Model 717-plus autosampler, a
Model 486 tunable absorbance detector, and a Model 600 controller) that
was equipped with a VYDAC.TM. C18 column (Supelco, Bellefonte, Pa.).
Proteins were dissolved in distilled, deionized water and filtered (0.22
.mu.m) prior to injection. Elution profiles were monitored at 215 nm with
a linear 2% gradient of water (0.1% trifluoroacetic acid) to acetonitrile
that was run over a period of 60 minutes at a flow rate of 1 ml/minute.
FIGS. 20A, 20B, and 20C are reverse-phase high performance liquid
chromatograms of genetically engineered proteins corresponding,
respectively, to A-Cys (76 .mu.g) eluted at 26.76 minutes, C.sub.10 -Trp
(54 .mu.g) eluted at 19.36 minutes, and AC.sub.10 A-Cys (54 .mu.g) eluted
at 27.33 minutes. Solvents were purged with helium. Amino acid
compositional analysis, as well as matrix-assisted laser-desorption mass
spectrometry (MALDI-MS), were carried out in the Analytical Chemistry and
Peptide/DNA Synthesis Facility at Cornell University.
Amino acid compositional analysis was made on a Pico-Tag Amino Acid
Analysis System (Millipore Corp., Bedford, Mass.). Amino acids were
derivatized with phenyl isothiocyanate (PITC) and analyzed by
reverse-phase HPLC with a 4.6.times.300 mm Nova Pack C18 column (Waters
Corp., Bedford, Mass.). All amino acid analyses were within 5% of the
theoretical values.
For MALDI experiments, protein samples were mixed with a molar excess of
matrix solution (0.05 M 3,5-dimethoxy-4-hydroxycinnamic acid for predicted
masses greater than 20,000 and 0.05 M .alpha.-cyano-4-hydroxycinnamic acid
for predicted masses less than 20,000 in 30% acetonitrile/water, 0.1%
trifluoroacetic acid). The probe surface was irradiated with a nitrogen
laser at 337 nm. Protein ions were recorded in the positive ion mode at a
laser power ranging from 10 to 25 kV. The calculated mass of A-Cys is
8549; a peak was found at 8548. Likewise, the calculated mass of C.sub.10
-Trp is 10383 and a peak was reported at 10433. The calculated mass of
AC.sub.10 A-Cys is 22439; the mass analysis gave a peak at 22478, probably
due to association of potassium counterion (molecular weight, 39).
Gelation through physical crosslinking requires association of the
coiled-coil elements of the block copolymers. To determine whether the
unfolding behavior of the helical blocks is affected by attachment of the
coil domain, the pH dependence of the transition temperature, T.sub.m, was
measured for both A-Cys (5 .mu.m) and AC.sub.10 A-Cys (5 .mu.m) in
solution. The similarity of the unfolding processes is shown in Table 5.
Wavelength scans of AC.sub.10 A-Cys showed a substantial helical
contribution to the secondary structure, indicated by minima at 208 nm and
222 nm, which are characteristic wavelengths for the .alpha.-helical
conformation.
TABLE 5
______________________________________
Protein pH T.sub.m (.degree. C.)
______________________________________
A-Cys 6.1 88
A-Cys 6.9 64
A-Cys 7.6 51
A-Cys 9.4 50
A-Cys 10.7 40
A-Cys 11.5 31
AC.sub.10 A-Cys 6.1 81
AC.sub.10 A-Cys 6.9 55
AC.sub.10 A-Cys 7.6 54
AC.sub.10 A-Cys 9.5 49
AC.sub.10 A-Cys 10.8 31
______________________________________
At pH values greater than 10, the unfolding transitions of both A-Cys and
AC.sub.10 A-Cys occur between 30 and 40.degree. C. The low T.sub.m is
expected in basic solution, in which the glutamic acid residues are
deprotonated and charge--charge repulsion of the helices will occur. In
addition, the helical content at 0.degree. C. is reduced from 22055 (pH
7.6) to 16835 (pH 11.5) deg cm.sup.2 dmol.sup.-1 for A-Cys and from 13722
(pH 7.6) to 10294 (10.8) deg cm.sup.2 dmol.sup.-1 for AC.sub.10 A-Cys.
This result suggests that the individual helices are partially unfolded at
highly basic conditions and low temperatures.
When the pH is lowered to values between 7 and 10, the T.sub.m values
increase slightly but are within 10.degree. C. of each other. In the range
of pH 7-10 and lower, the lysine (pKa=10) is charged and can form
interhelical salt bridges with the glutamic acid. Upon further decrease of
the pH to 6, the T.sub.m for A-Cys and AC.sub.10 A-Cys occurs at
88.degree. C. and 81.degree. C., respectively. At pH 5 and lower, no
transitions are observed for either protein at temperatures below
100.degree. C. and the helical content decreases only slightly throughout
the 0-100.degree. C. temperature range. Acidic conditions effectively
stabilize the helix so that no thermal denaturation of A-cys or AC.sub.10
A-cys is observed.
The thermal denaturation experiments described above were carried out in
solutions containing 150 mM sodium chloride. Although the addition of
sodium chloride (>100 mM) had no significant effect on coiled-coil
stability at neutral pH, the stability of the coiled-coil increased
drastically in acidic environments. Similar behavior was observed for the
helical domains in this study. A thermal melting temperature, T.sub.m, was
observed for protein A-Cys at 85.degree. C. in 10 mM dibasic sodium
phosphate, pH 2.1 (without sodium chloride). No thermal transitions at 222
nm are observed from 0 to 100.degree. C. for either A-Cys or AC.sub.10
A-Cys at pH 2.1. The lack of a thermal transition below 100.degree. C.
suggested that the helices were stabilized by the addition of sodium
chloride in acidic environments.
The thermal dependence of gelation for AC.sub.10 A-Cys was recorded by
cycling a 5% gel through a series of temperatures ranging from 23.degree.
C. to 55.degree. C. at pH 7.8. The average mean square displacement
plotted against time at various temperatures is shown in the graph in FIG.
21. These data were acquired in the following temperature order: 23, 39.6,
47.7, 55.2, 42.7, 24.9, and 23.degree. C. (5% w/v, 10 mM Tris buffer, pH
7.8). In FIG. 21, data collected upon heating the sample from 23.degree.
C. to 55.2.degree. C. is represented by (a) and the data collected on
cooling the sample from 55.2.degree. C. back down to 23.degree. C. is
represented by (b).
These data show that as the temperature was increased to 55.degree. C., the
gel became more fluid. Interestingly, hysteresis was observed after the
material was heated to 55.degree. C. and returned to 23.degree. C. (see
FIG. 22, which shows the mean square displacement before, a, and after, b,
thermal cycling). This indicates either that equilibrium was not reached
or that the gel did not recover 100%.
The dependence of gel formation on concentration was also investigated for
AC.sub.10 A-Cys at pH 7.9 by diffusing wave spectroscopy (DWS). The DWS
spectrum revealed that AC.sub.10 A-Cys became a fluid at 3% (w/v), but
AC.sub.10 A-Cys was an elastic medium at 5% (FIG. 23). The 4% sample
formed a viscous liquid, at the time of preparation, but the emergence of
a plateau was apparent at intermediate times. Therefore, the critical
concentration for forming an elastic gel spanning the entire volume of
interest was 4-5%. At concentrations of 3-4%, the protein was most likely
forming soluble aggregates that viscosified the solution by increasing the
apparent molecular weight.
Example 3
Incorporation of 5',5',5'-Trifluoro-L-leucine at the Hydrophobic Interface
of Leucine Zippers
Leucine auxotrophs of strain MC1000 (F-lac.DELTA.x74anaD139/(Ana
Abioc-leu).DELTA.7679 gal U gal K rspL) containing pREP4 plasmid (Qiagen,
Chatsworth, Calif.) were used. The stable auxotroph was designated JM-1.
Expression vectors containing A1 and B1 DNA sequences (pQE9-A1 and
pQE9-B1) were used.
General Methods
Electrospray mass spectra were collected on a FISONS VG PLATFORM II.TM.
electrospray mass spectrometer. A1 and B1 protein spectra were recorded as
standards. All samples were run in 50:50 acetonitrile:water with 0.2%
formic acid at 12 pmol/.mu.l concentrations. Data were recorded in a
positive ion mode (full scan 600-1500 m/z), with an infusion rate of 5
.mu.l/minute and cone voltages of 35 and 50 V.
A Waters high performance liquid chromatography (HPLC) system (including a
Model 717-plus autosampler, a Model 486 tunable absorbance detector, and a
Model 600 controller) was used to inject 50 .mu.l samples at flow rates of
1 ml/min onto a VYDAC.TM. C18 (Supelco, Bellefonte, Pa.) reversed phase
column. MILLENNIUM.TM. (Waters Corp., Milford, Mass.) software was used in
the analysis of the collected spectra at 210 nm. The samples were eluted
over 60 minutes with a gradient of water/0.1% trifluoroacetic acid (TFA)
and acetonitrile/0.1% TFA. The gradient included 90:10 (10 min), 50:50 (20
min), and 100:0 (30 min) ratios. Amino acid analysis was carried out on a
PICO-TAG.TM. Amino Acid Analysis System. Duplicate samples were hydrolyzed
in constant boiling hydrochloric acid at 150.degree. C. for 110 minutes.
Appropriate blanks, controls and standards were hydrolyzed in the same
vessel as a batch hydrolysis. The amino acids were derivatized with
phenylisothiocyanate (PITC) and analyzed by reverse-phase HPLC. The
resulting phenylthiocarbamoyl amino acid derivatives were separated on a
4.6.times.300 mm Nova Pack C18 column, using a modified Pico-Tag buffer
system. Circular dichroism spectra were recorded on an Aviv 62DS
spectropolarimeter (Lakewood, N.J.). Mean residue molar ellipticity
reported at 222 nm ([.theta.].sub.222, deg cm.sup.2 dmol.sup.-1) was
calculated from the following equation:
[.theta.]=[.theta.].sub.obs .times.MRW/(c.times.1)
where [.theta.].sub.obs is ellipticity measured in millidegrees, MRW is the
mean residue molecular weight (i.e., molecular weight of the peptide
divided by the number of amino acid residues), c is the peptide
concentration in g/l determined by quantitative amino acid analysis, and 1
is the optical path length in mm. Thermal melting curves were determined
by monitoring the CD signal of the protein solutions (3 .mu.M) at 222 nm
as a function of temperature. Data was collected from 0 to 90.degree. C.
in 1.degree. C. increments with an equilibration time of 1 minute. Spectra
were collected in 50 mM sodium phosphate buffer, 100 mM sodium chloride,
pH 7.4. Two Peltier units on both sides of the 1 cm path length
rectangular cell (Helma) were used to regulate the temperature to within
.+-.0.2.degree. C.
Thermal melting temperature, T.sub.m, was calculated as the temperature at
which 50% of the helix is unfolded, using a two-state mechanism to
describe the equilibrium between folded and unfolded protein states. To
get this value, ellipticity readings were normalized to the fraction of
the protein folded using the standard equation:
f.sub.n =([.theta.]-[.theta.].sub.u)/([.theta.].sub.n -[.theta.].sub.u)
where [.theta.].sub.n and [.theta.].sub.u represent the ellipticity values
for the fully folded and fully unfolded species, respectively, at 222 nm.
All thermal melting transitions were determined to be reversible
(.+-.2.degree. C.) with 96%, 88%, 99%, and 97% of the initial helix
contents of A1, B1, Tfl A1, and Tfl B1, respectively.
Protein Expression and Purification
pQE9-A1 and pQE9-B1 were used to transform strain mc1000
(F-lac.DELTA.x74anaD139/(Ana Abioc-leu).DELTA.7679 gal U gal K rspL)
containing pREP4 to yield JM-1/pQE9-A1 and JM-1/pQE9-B1. Single colonies
of cells were grown to saturation in 5 ml of 2xYT medium (16 g
Bacto-Tryptone, 10 g yeast extract, 5 g sodium chloride per liter)
containing ampicillin (200 .mu.g/ml) and kanamycin (25 .mu.g/ml) overnight
at 37.degree. C. Each of these cultures was transferred to 1 l of
sterilized YT medium containing the same concentrations of antibiotics.
The cells were incubated until optical densities at 600 nm (OD.sub.600)
reached 1 and then centrifuged at 5000 rpm for 10 minutes. The supernatant
was then decanted and the cells were washed with 1 l of 1% M9 salt
solution (containing 60 g dibasic sodium phosphate, 30 g monobasic
potassium phosphate, 5 g sodium chloride, and 10 g ammonium chloride per
liter). Again the cells were pelleted and resuspended in 2 l of M9 medium
(containing 10% v/v M9 salt solution, 10% 19 amino acid solution (0.4 g of
each amino acid, except leucine, per liter), 0.1% 1 M magnesium sulfate
heptahydrate, 0.1% 0.01 M calcium chloride, 0.1% vitamin B.sub.1, 4%
glucose (20% w/v solution), 0.33% ampicillin (200 .mu.g/ml), 0.16%
kanamycin (25 .mu.g/ml) and 75.3% distilled deionized water). Leucine (40
mg/ml; final concentration of 40 .mu.g/ml) or
5',5',5'-trifluoro-D,L-leucine (40 mg/ml; final concentration of 40
.mu.g/ml; MTM Research Chemicals, Windham, N.H.) was added in addition to
the M9 medium. The cells were grown for 15 minutes, then
isopropyl-.beta.-thiogalactoside (IPTG, 1 mM) was added to induce protein
synthesis. Cell cultures were removed and centrifuged at 5000 rpm for 10
minutes at various times after induction with IPTG. All pelleted cells
were resuspended in 25 ml of lysis buffer (6 M guanidine-HCl, 0.1 M
dibasic sodium phosphate, pH 8) and placed in -80.degree. C. freezer
overnight. The supernatants from the cell lysates were subjected to
nickel(II)-NTA metal affinity chromatography. The collected protein
fractions were frozen at -80.degree. C. and lyophilized to give total dry
weights for all proteins of 75-100 mg per liter of growth medium.
The in vivo synthesis of Tfl A1 and Tfl B1 was followed after the culture
was shifted to minimal medium supplemented with amino acids. Protein
accumulation was monitored for three hours after induction with IPTG (1
mM) and was visualized on a 15% SDS-polyacrylamide gel with Coomassie
Brilliant Blue R-250. Prior to induction there was no protein
accumulation; one to three hours after induction, cells in medium
containing either Leu or Tfl showed protein expression. Substitution of
Tfl for leucine in A1 and B1 was confirmed by electrospray mass
spectrometry.
Initial measurements of the thermal stabilities of .alpha.-helical Leu and
Tfl containing A1 and B1 were made by circular dichroism spectroscopy
(Table 6 and FIGS. 24A and 24B). Table 6 lists the thermal melting
transitions of A1, Tfl A1, B1, and Tfl B1, monitored at 222 nm; T.sub.m is
the temperature at which 50% of the helix is unfolded in 50 mM sodium
phosphate, 100 mM sodium chloride, pH 7.4.
The thermal stabilities of the Tfl-substituted helices recorded at 222 nm
showed that 66% replacement of leucine with trifluoroleucine in A1 only
resulted in an 8.degree. C. increase in T.sub.m, whereas 38% replacement
in B1 resulted in a 13.degree. C. increase in T.sub.m (the spectra were
acquired in 50 mM dibasic sodium phosphate and 100 mM sodium chloride, pH
7.4,
______________________________________
Dimer Complex T.sub.m in PBS (.degree. C.).sup.a
______________________________________
A1--A1 58
Tfl A1--Tfl A1 66
B1--B1 75
Tfl B1--Tfl B1 88
______________________________________
at protein concentrations of 3 .mu.M). These data suggested that the
increased level of Tfl destabilized the helix--helix interactions due to:
(1) the increased size of fluorine over that of hydrogen, (2) the
difference in chemical character between fluorine and hydrogen, or (3)
both the differences in size and character. The introduction of Tfl at the
d position in the heptad repeat can disrupt the overall packing of
coiled-coils.
At lower levels of Tfl substitution, the effect of increased size and
electronegativity on the packing density can be less significant than at
higher levels. The interaction between fluorine and hydrogen is relatively
unfavorable; thus, at intermediate levels of Tfl substitution, the helices
are destablized. At near complete Tfl substitution at the d position,
however, it is thought the helices would be very stable due to favorable
fluorine--fluorine interaction.
In general, the results demonstrate that coiled-coils became more thermally
stable with relatively low levels of Tfl substitution. The use of leucine
auxotrophs to prepare unique coiled-coil proteins containing Tfl groups
can be applied to a variety of proteins where Leu participates in folding,
stability, activity, or function. With optimization of this strategy, the
enhancement of certain physical properties in natural polymer systems can
be achieved for biological or materials applications.
Example 4
Use of a Hydrogel as a Wound Dressing
A solution is made from the AC.sub.10 A copolymer. This solution forms a
stable gel at around pH 7 over a broad range of temperatures (i.e.,
including at least 25 to 37.degree. C.) as a result of the interaction and
packing of the coiled-coil structure. The pH of the gelled solution is
increased to 12, at which the solution liquifies. A topical antibiotic
drug (e.g., neosporin) is dissolved in the solution and loaded into an
aerosol can for later use.
A brief burst of the aerosol is used to dress a wound on a patient (e.g.,
an abrasion, burn, or non-puncture wound). When the aerosol contacts the
patient's skin, the pH of the solution is partly neutralized by water,
sweat, or blood on the skin's surface, thereby causing the copolymer to
form a protective medicated gel covering the wound.
Example 5
Use of a Copolymer to Promote Healing
A copolymer is produced from a gene that encodes a derivative of AC.sub.10
A with a cell binding domain inserted in the middle of the random coil
block. The cell binding domain is the integrin ArgGlyAspSer SEQ ID NO: 22
sequence, which is known to bind to gpIIa/IIIa proteins expressed on
fibroblast cells necessary for matrix formation for the regeneration of
skin. The AC.sub.10 A protein is chosen because it is a gel under
physiological conditions (i.e., pH 7.4, 37.degree. C.). The produced
copolymer is suspended in water to form a gel, which is then is used to
treat a wound. Fibroblasts become entrapped within the gel and thus remain
at the site of the wound, serving as a scaffold for the regeneration of
tissue surrounding the wound.
Other Embodiments
It is to be understood that while the invention has been described in
conjunction with the detailed description thereof, the foregoing
description is intended to illustrate and not limit the scope of the
invention, which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
__________________________________________________________________________
# SEQUENCE LISTING
- (1) GENERAL INFORMATION:
- (iii) NUMBER OF SEQUENCES: 24
- (2) INFORMATION FOR SEQ ID NO:1:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 42 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
- Ser Gly Asp Leu Glu Asn Glu Val Ala Gln Le - #u Glu Arg Glu Val Arg
# 15
- Ser Leu Glu Asp Glu Ala Ala Glu Leu Glu Gl - #n Lys Val Ser Arg Leu
# 30
- Lys Asn Glu Ile Glu Asp Leu Lys Ala Glu
# 40
- (2) INFORMATION FOR SEQ ID NO:2:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 42 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
- Ser Gly Asp Leu Lys Asn Lys Val Ala Gln Le - #u Lys Arg Lys Val Arg
# 15
- Ser Leu Lys Asp Lys Ala Ala Glu Leu Lys Gl - #n Glu Val Ser Arg Leu
# 30
- Glu Asn Glu Ile Glu Asp Leu Lys Ala Lys
# 40
- (2) INFORMATION FOR SEQ ID NO:3:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 14 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
- Met Arg Gly Ser His His His His His His Gl - #y Ser Met Ala
# 10
- (2) INFORMATION FOR SEQ ID NO:4:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 18 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
- Ile Gly Asp Leu Asn Asn Thr Ser Gly Ile Ar - #g Arg Pro Ala Ala Lys
# 15
- Leu Asn
- (2) INFORMATION FOR SEQ ID NO:5:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 77 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
- AATCGGATCC GATGACGATG ACAAAGCTAG CTATCGCGAT GGTGACCCGC GC - #ATGCCGAC
60
# 77 A
- (2) INFORMATION FOR SEQ ID NO:6:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 78 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
- AGCTTGGATC CTTACCAACT AGTCGGCATG CGCGGGTCAC CATCGCGATA GC - #TAGCTTTG
60
# 78 CG
- (2) INFORMATION FOR SEQ ID NO:7:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 86 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
- AATTCGGATC CGATGACGAT GACAAATGGG CTAGCGGTGA CCATGTGGCG CC - #TCGAGACA
60
# 86 CTAG GATCCA
- (2) INFORMATION FOR SEQ ID NO:8:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 86 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
- AGCTTGGATC CTAGCAGCCA CCCATACTAG TGTCTCGAGG CGCCACATGG TC - #ACCGCTAG
60
# 86 ATCG GATCCG
- (2) INFORMATION FOR SEQ ID NO:9:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 292 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
- CGATCCGATG GGTGCCGGCG CTGGTGCGGG CCCGGAAGGT GCAGGCGCTG GT - #GCGGGCCC
60
- GGAAGGTGCC GGCGCTGGTG CGGGCGGCGA AGGTGCAGGC GCTGGTGCGG GC - #CCGGAAGG
120
- TGCCGGCGCT GGTGCGGGCC CGGAAGGTGC AGGCGCTGGT GCGGGCCCGG AA - #GGTGCCGG
180
- CGCTGGTGCG GGCCCGGAAG GTGCAGGCGC TGGTGCGGGC CCGGAAGGTG CC - #GGCGCTGG
240
- TGCGGGCCCG GAAGGTGCAG GCGCTGGTGC GGGCCCGGAA GGTGCCCGCA TG - #
292
- (2) INFORMATION FOR SEQ ID NO:10:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 288 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
- CGGGCACCTT CCGGGCCCGC ACCAGCGCCT GCACCTTCCG GGCCCGCACC AG - #CGCCGGCA
60
- CCTTCCGGGC CCGCACCAGC GCCTGCACCT TCCGGGCCCG CACCAGCGCC GG - #CACCTTCC
120
- GGGCCCGCAC CAGCGCCTGC ACCTTCCGGG CCCGCACCAG CGCCGGCACC TT - #CCGGGCCC
180
- GCACCAGCGC CTGCACCTTC GCCGCCCGCA CCAGCGCCGG CACCTTCCGG GC - #CCGCACCA
240
# 288GGCC CGCACCAGCG CCGGCACCCA TCGGATCG
- (2) INFORMATION FOR SEQ ID NO:11:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 778 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
- CGATCCGATG GGTGCCGGCG CTGGTGCGGG CCCGGAAGGT GCAGGCGCTG GT - #GCGGGCCC
60
- GGAAGGTGCC GGCGCTGGTG CGGGCGGCGA AGGTGCAGGC GCTGGTGCGG GC - #CCGGAAGG
120
- TGCCGGCGCT GGTGCGGGCC CGGAAGGTGC AGGCGCTGGT GCGGGCCCGG AA - #GGTGCCGG
180
- CGCTGGTGCG GGCCCGGAAG GTGCAGGCGC TGGTGCGGGC CCGGAAGGTG CC - #GGCGCTGG
240
- TGCGGGCCCG GAAGGTGCAG GCGCTGGTGC GGGCCCGGAA GGTGCCGGCG CT - #GGTGCGGG
300
- CCCGGAAGGT GCAGGCGCTG GTGCGGGCCC GGAAGGTGCC GGCGCTGGTG CG - #GGCCCGGA
360
- AGGTGCAGGC GCTGGTGCGG GCCCGGAAGG TGCCGGCGCT GGTGCGGGCC CG - #GAAGGTGC
420
- AGGCGCTGGT GCGGGCCCGG AAGGTGCCGG CGCTGGTGCG GGCCCGGAAG GT - #GCAGGCGC
480
- TGGTGCGGGC CCGGAAGGTG CCGGCGCTGG TGCGGGCCCG GAAGGTGCAG GC - #GCTGGTGC
540
- GGGCCCGGAA GGTGCCGGCG CTGGTGCGGG CCCGGAAGGT GCAGGCGCTG GT - #GCGGGCCC
600
- GGAAGGTGCC GGCGCTGGTG CGGGCCCGGA AGGTGCAGGC GCTGGTGCGG GC - #CCGGAAGG
660
- TGCCGGCGCT GGTGCGGGCC CGGAAGGTGC AGGCGCTGGT GCGGGCCCGG AA - #GGTGCCGG
720
- CGCTGGTGCG GGCCCGGAAG GTGCAGGCGC TGGTGCGGGC CCGGAAGGTG CC - #CGCATG
778
- (2) INFORMATION FOR SEQ ID NO:12:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 774 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
- CGGGCACCTT CCGGGCCCGC ACCAGCGCCT GCACCTTCCG GGCCCGCACC AG - #CGCCGGCA
60
- CCTTCCGGGC CCGCACCAGC GCCTGCACCT TCCGGGCCCG CACCAGCGCC GG - #CACCTTCC
120
- GGGCCCGCAC CAGCGCCTGC ACCTTCCGGG CCCGCACCAG CGCCGGCACC TT - #CCGGGCCC
180
- GCACCAGCGC CTGCACCTTC CGGGCCCGCA CCAGCGCCGG CACCTTCCGG GC - #CCGCACCA
240
- GCGCCTGCAC CTTCCGGGCC CGCACCAGCG CCGGCACCTT CCGGGCCCGC AC - #CAGCGCCT
300
- GCACCTTCCG GGCCCGCACC AGCGCCGGCA CCTTCCGGGC CCGCACCAGC GC - #CTGCACCT
360
- TCCGGGCCCG CACCAGCGCC GGCACCTTCC GGGCCCGCAC CAGCGCCTGC AC - #CTTCCGGG
420
- CCCGCACCAG CGCCGGCACC TTCCGGGCCC GCACCAGCGC CTGCACCTTC CG - #GGCCCGCA
480
- CCAGCGCCGG CACCTTCCGG GCCCGCACCA GCGCCTGCAC CTTCCGGGCC CG - #CACCAGCG
540
- CCGGCACCTT CCGGGCCCGC ACCAGCGCCT GCACCTTCCG GGCCCGCACC AG - #CGCCGGCA
600
- CCTTCCGGGC CCGCACCAGC GCCTGCACCT TCCGGGCCCG CACCAGCGCC GG - #CACCTTCC
660
- GGGCCCGCAC CAGCGCCTGC ACCTTCGCCG CCCGCACCAG CGCCGGCACC TT - #CCGGGCCC
720
- GCACCAGCGC CTGCACCTTC CGGGCCCGCA CCAGCGCCGG CACCCATCGG AT - #CG
774
- (2) INFORMATION FOR SEQ ID NO:13:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 126 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
- GTGACCTGGA AAACGAAGTG GCCCAGCTGG GAAGGGAAGT TAGATCTCTG GA - #AGATGAAG
60
- CGGCTGAACT GGAACAAAAA GTCTCGAGAC TGAAAAATGA AATCGAAGAC CT - #GAAAGCCG
120
# 126
- (2) INFORMATION FOR SEQ ID NO:14:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 126 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
- GTCACCAATT TCGGCTTTCA GGTCTTCGAT TTCATTTTTC AGTCTCGAGA CT - #TTTTGTTC
60
- CAGTTCAGCC GCTTCATCTT CCAGAGATCT AACTTCCCTT TCCAGCTGGG CC - #ACTTCGTT
120
# 126
- (2) INFORMATION FOR SEQ ID NO:15:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 126 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
- GTGACCTGAA AAACAAAGTG GCCCAGCTGA AAAGGAAAGT TAGATCTCTG AA - #AGATAAAG
60
- CGGCTGAACT GAAACAAGAA GTCTCGAGAC TGGAAAATGA AATCGAAGAC CT - #GAAAGCCA
120
# 126
- (2) INFORMATION FOR SEQ ID NO:16:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 126 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
- GTCACCAATT TTGGCTTTCA GGTCTTCGAT TTCATTTTCC AGTCTCGAGA CT - #TCTTGTTT
60
- CAGTTCAGCC GCTTTATCTT TCAGAGATCT AACTTTCCTT TTCAGCTGGG CC - #ACTTTGTT
120
# 126
- (2) INFORMATION FOR SEQ ID NO:17:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 76 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
- Met Arg Gly Ser His His His His His His Gl - #y Ser Asp Asp Asp Asp
# 15
- Lys Trp Ala Ser Gly Asp Leu Glu Asn Glu Va - #l Ala Gln Leu Glu Arg
# 30
- Glu Val Arg Ser Leu Glu Asp Glu Ala Ala Gl - #u Leu Glu Gln Lys Val
# 45
- Ser Arg Leu Lys Asn Glu Ile Glu Asp Leu Ly - #s Ala Glu Ile Gly Asp
# 60
- His Val Ala Pro Arg Asp Thr Ser Met Gly Gl - #y Cys
# 75
- (2) INFORMATION FOR SEQ ID NO:18:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 76 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
- Met Arg Gly Ser His His His His His His Gl - #y Ser Asp Asp Asp Asp
# 15
- Lys Trp Ala Ser Gly Asp Leu Lys Asn Lys Va - #l Ala Gln Leu Lys Arg
# 30
- Lys Val Arg Ser Leu Lys Asp Lys Ala Ala Gl - #u Leu Lys Gln Glu Val
# 45
- Ser Arg Leu Lys Asn Glu Ile Glu Asp Leu Ly - #s Ala Lys Ile Gly Asp
# 60
- His Val Ala Pro Arg Asp Thr Ser Met Gly Gl - #y Cys
# 75
- (2) INFORMATION FOR SEQ ID NO:19:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 122 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
- Met Arg Gly Ser His His His His His His Gl - #y Ser Asp Asp Asp Asp
# 15
- Lys Ala Ser Tyr Arg Asp Pro Met Gly Ala Gl - #y Ala Gly Ala Gly Pro
# 30
- Glu Gly Ala Gly Ala Gly Ala Gly Pro Glu Gl - #y Ala Gly Ala Gly Ala
# 45
- Gly Pro Glu Gly Ala Gly Ala Gly Ala Gly Pr - #o Glu Gly Ala Gly Ala
# 60
- Gly Ala Gly Pro Glu Gly Ala Gly Ala Gly Al - #a Gly Pro Glu Gly Ala
# 80
- Gly Ala Gly Ala Gly Pro Glu Gly Ala Gly Al - #a Gly Ala Gly Pro Glu
# 95
- Gly Ala Gly Ala Gly Ala Gly Pro Glu Gly Al - #a Gly Ala Gly Ala Gly
# 110
- Pro Glu Gly Ala Arg Met Pro Thr Ser Trp
# 120
- (2) INFORMATION FOR SEQ ID NO:20:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 123 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
- GGTGACCTGA AAAACAAAGT GGCCCAGCTG AAAAGCAAAG TTAGATCTCT GA - #AAGATAAA
60
- GCGGCTGAAC TGAAACAAGA AGTCTCGAGA CTGGAAAATG AAATCGAAGA CC - #TGAAAGCC
120
# 123
- (2) INFORMATION FOR SEQ ID NO:21:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 123 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
- GGTGACCTGG AAAACGAAGT GGCCCAGCTG GGAAGGGAAG TTAGATCTCT GG - #AAGATGAA
60
- GCGGCTGAAC TGGAACAAAA AGTCTCGAGA CTGAAAAATG AAATCGAAGA CC - #TGAAAGCC
120
# 123
- (2) INFORMATION FOR SEQ ID NO:22:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 4 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
- Arg Gly Asp Ser
1
- (2) INFORMATION FOR SEQ ID NO:23:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 308 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: Protein
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 1...8
#where any of the first set starting at
#1 through 8 may be absent or present
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 9...20
#where amino acids shown atTION:
#9 through 20 are Pro Glu Gly
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 21...308
#where any of the subsets starting at
#21 through 308 may be absent or present
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
- Ala Gly Ala Gly Ala Gly Ala Gly Pro Glu Gl - #y Pro Glu Gly Pro Glu
# 15
- Gly Pro Glu Gly Pro Glu Gly Pro Glu Gly Pr - #o Glu Gly Pro Glu Gly
# 30
- Pro Glu Gly Pro Glu Gly Pro Glu Gly Pro Gl - #u Gly Pro Glu Gly Pro
# 45
- Glu Gly Pro Glu Gly Pro Glu Gly Pro Glu Gl - #y Pro Glu Gly Pro Glu
# 60
- Gly Pro Glu Gly Pro Glu Gly Pro Glu Gly Pr - #o Glu Gly Pro Glu Gly
# 80
- Pro Glu Gly Pro Glu Gly Pro Glu Gly Pro Gl - #u Gly Pro Glu Gly Pro
# 95
- Glu Gly Pro Glu Gly Pro Glu Gly Pro Glu Gl - #y Pro Glu Gly Pro Glu
# 110
- Gly Pro Glu Gly Pro Glu Gly Pro Glu Gly Pr - #o Glu Gly Pro Glu Gly
# 125
- Pro Glu Gly Pro Glu Gly Pro Glu Gly Pro Gl - #u Gly Pro Glu Gly Pro
# 140
- Glu Gly Pro Glu Gly Pro Glu Gly Pro Glu Gl - #y Pro Glu Gly Pro Glu
145 1 - #50 1 - #55 1 -
#60
- Gly Pro Glu Gly Pro Glu Gly Pro Glu Gly Pr - #o Glu Gly Pro Glu Gly
# 175
- Pro Glu Gly Pro Glu Gly Pro Glu Gly Pro Gl - #u Gly Pro Glu Gly Pro
# 190
- Glu Gly Pro Glu Gly Pro Glu Gly Pro Glu Gl - #y Pro Glu Gly Pro Glu
# 205
- Gly Pro Glu Gly Pro Glu Gly Pro Glu Gly Pr - #o Glu Gly Pro Glu Gly
# 220
- Pro Glu Gly Pro Glu Gly Pro Glu Gly Pro Gl - #u Gly Pro Glu Gly Pro
225 2 - #30 2 - #35 2 -
#40
- Glu Gly Pro Glu Gly Pro Glu Gly Pro Glu Gl - #y Pro Glu Gly Pro Glu
# 255
- Gly Pro Glu Gly Pro Glu Gly Pro Glu Gly Pr - #o Glu Gly Pro Glu Gly
# 270
- Pro Glu Gly Pro Glu Gly Pro Glu Gly Pro Gl - #u Gly Pro Glu Gly Pro
# 285
- Glu Gly Pro Glu Gly Pro Glu Gly Pro Glu Gl - #y Pro Glu Gly Pro Glu
# 300
- Gly Pro Glu Gly
305
- (2) INFORMATION FOR SEQ ID NO:24:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 700 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: Protein
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 1...22
#where Xaa at positions 1, 8, 15, and 22
#any one of Ala, Gly, Ile, Leu, Met, Phe, Pro - #, Trp,
or Val
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 2...23
#where Xaa at positions 2, 9, 16, and 23
#any amino acidmay be
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 3...24
#where Xaa at positions 3, 10, 17, and 24
#any amino acidmay be
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 4...25
#where Xaa at positions 4, 11, 18, and 25
#any one of Ala, Gly, Ile, Leu, Met, Phe, Pro - #, Trp,
or Val
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 5...26
#where Xaa at positions 5, 12, 19, and 26
#any one of Asn, Cys, Gln, Thr, Val, Lys, His - #, Glu,
Asp, Arg, - # or Ser
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 6...27
#where Xaa at positions 6, 13, 20, and 27
#any amino acidmay be
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 7...28
#where Xaa at positions 7, 14, 21, and 28
#any one of Asn, Cys, Gln, Thr, Val, Lys, His - #, Glu,
Asp, Arg, - # or Ser
- (ix) FEATURE:
(A) NAME/KEY: Other
(B) LOCATION: 29...308
#where Xaa in any subset from positions 29
through 3 - #08 may be absent or present
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 15
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 30
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 45
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 60
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 80
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 95
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 110
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 125
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 140
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
145 1 - #50 1 - #55 1 -
#60
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 175
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 190
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 205
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 220
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
225 2 - #30 2 - #35 2 -
#40
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 255
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 270
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 285
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 300
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
305 3 - #10 3 - #15 3 -
#20
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 335
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 350
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 365
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 380
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
385 3 - #90 3 - #95 4 -
#00
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 415
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 430
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 445
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 460
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
465 4 - #70 4 - #75 4 -
#80
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 495
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 510
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 525
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 540
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
545 5 - #50 5 - #55 5 -
#60
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 575
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 590
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 605
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 620
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
625 6 - #30 6 - #35 6 -
#40
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 655
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 670
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 685
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa
# 700
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